Production of human lactoferrin in animal milk.

Genetic constructs containing the human lactoferrin (hLf) gene were created within a joint program of Russian and Belorussian scientists. Using these constructs, transgenic mice were bred (the maximum hLf concentration in their milk was 160 g/L), and transgenic goats were also generated (up to 10 g/L hLf in their milk). Experimental goatherds that produced hLf in their milk were also bred, and the recombinant hLf was found to be identical to the natural protein in its physical and chemical properties. These properties included electrophoretic mobility, isoelectric point, recognition by polyclonal and monoclonal antibodies, circular dichroic spectra, interaction with natural ligands (DNA, lipopolysaccharides, and heparin), the binding of iron ions, the sequence of the 7 terminal amino acids, and its biological activity. The latter was assessed by the agglutination of Micrococcus luteus protoplasts, bactericidal activity against Escherichia coli and Listeria monocytogenes , and fungicidal activity against Candida albicans . We also demonstrated a significant increase in the activity of antibiotics when used in combination with Lf.

Expression of Sambucus nigra agglutinin (SNA-I') from elderberry bark in transgenic tobacco plants results in enhanced resistance to different insect species.

Tobacco plants (Nicotiana tabacum cv Samsun NN) have been transformed with the gene encoding the type-2 ribosome-inactivating protein (RIP) SNA-I' from elderberry (Sambucus nigra) under the control of the Cauliflower Mosaic Virus 35S promoter. Previous research confirmed that these plants synthesize, correctly process and assemble a fully active RIP. Variability in protein expression was observed within the transgenic lines. The effects of the type-2 RIP SNA-I' delivered through a leaf feeding assay were evaluated in the laboratory on two economically important pest insects belonging to the orders of Hemiptera, the tobacco aphid (Myzus nicotianae) and Lepidoptera, the beet armyworm (Spodoptera exigua). In the experiment with aphids, significant effects were observed on the life parameters, such as survival, intrinsic rate of increase, net reproductive rate, mean generation time and mean daily offspring, whereas with caterpillars significant reduction in fresh weight as well as retardation in development were observed. In addition, significant increases in mortality were noted for insects fed on the transgenic lines as compared to wild type plants. This information provides further support for RIPs having a role in plant resistance to insect pest species.

Cold agglutinin syndrome in pediatric liver transplant recipients.

Anemia is a common finding in post-liver transplant patients. Causes for the anemia include nutritional deficiencies, red cell aplasia as well as immune-mediated hemolysis. One of the immunologic causes of hemolytic anemia is drug-induced hemolysis. Tacrolimus is a common immunosuppressant used in post-liver transplant patients to prevent graft rejection. There have been reports of tacrolimus-associated hemolytic anemia secondary to hemolytic uremic syndrome as well as autoimmune hemolysis. There are also case-reports of severe hemolytic anemia related to cold agglutinin production in post-liver transplant patients. We described in this paper three cases of severe cold agglutinin hemolytic anemia in three pediatric liver transplant patients. Steroid therapy, plasmapheresis and withdrawal of tacrolimus led to resolution of the severe hemolytic process in each case. Whether the immune-mediated hemolysis is related to tacrolimus is not clear and needs to be characterized further.

B-lymphocytes as targets for therapy in chronic cold agglutinin disease.

Primary chronic cold agglutinin disease (CAD) is an autoimmune hemolytic anemia induced by cold reactive autoantibodies (cold agglutinins) against erythrocyte surface antigens. Corticosteroids or alkylating agents have been used in the treatment of CAD, but the results have been disappointing. The cold agglutinins in CAD patients are monoclonal immunoglobulins, usually of the IgMkappa type encoded by the V(H)4-34 gene segment. Flowcytometric assessment of lymphocytes from bone marrow aspirates and immunohistochemical assessment of biopsy samples have revealed a monoclonal CD20(+) kappa(+) B lymphocyte population in 90% of the patients. These pathogenetic features have provided a basis for novel therapies in primary CAD. Infusions of rituximab, a chimeric human-murine anti-CD20 antibody known to be effective in B-cell lymphoma, produced partial response rates of approximately 50% and occasional complete responses. Median response duration, however, was only 11 months. Complement C3 and C4 depletion in many CAD patients, as well as Fc-gamma-RIIIa receptor polymorphism, have been proposed as explanations for the inconstant efficacy of rituximab therapy. In order to increase response rates and response duration, we are undertaking a phase 2 study of rituximab and fludarabine combination therapy. The preliminary results are encouraging, but further studies are required in order to allow firm conclusions.

Expression and purification of Arisaema heterophyllum agglutinin in Escherichia coli.

Recombinant Arisaema heterophyllum agglutinin (AHA) was expressed in Escherichia coli as N-terminal His-tagged fusions. After induction with isopropylthio-beta-D-galactoside, the recombinant AHA was purified by metal-affinity chromatography. The purified AHA protein was incorporated into artificial diet at 0.1% (w/v) concentration in insect bioassay trial and the result showed that artificial diet containing AHA could significantly inhibit the growth of the third-instar nymphs of peach potato aphid (Myzus persicae). This study suggested that AHA could be an effective candidate for the control of peach potato aphid, one of the most serious sap-sucking insect pests causing significant yield loss of crops.

Induction of DMBT1 expression by reduced ERK activity during a gastric mucosa differentiation-like process and its association with human gastric cancer.

Abnormalities in the expression of DMBT1 (deleted in malignant brain tumors 1) have been implicated in the development of esophageal, gastric and colorectal cancers of the alimentary tract, but the underlying mechanism remains unclear. In the present study, using the gastric cell line AGS, we identified two intracellular signaling molecules protein kinase C (PKC) and extracellular signal-related kinase (ERK). They mediated both the phorbol myristate acetate (PMA) downregulation of DMBT1 expression and the initiation of cell differentiation, which was measured by cell cycle withdrawal and the induction of the tissue-specific marker trefoil factor 1 (TFF1). A time-course study showed that following the PMA activation of ERK kinase, the induction of TFF1 and the reduction of DMBT1 were detected at the same time point. We then demonstrated a minimal level of DMBT1 in proliferating AGS cells seeded at low density, where ERK activity was high. Reduction of ERK activity, either by an ERK inhibitor PD98059 or by high-density seeding, significantly reduced AGS cell growth judged by CFSE labeling. This cellular effect was elicited by cyclin D/p21 (Cip/Waf1) and G(0)/G(1) arrest, and was accompanied by a marked increase in DMBT1-expressing cells. Finally, we showed that siRNA directed against DMBT1 had no effect on the induction of a cell growth arrest marker, gut-enriched Kruppel-like factor (GKLF), but reduced the PMA induction of TFF1. Along with its upregulation coinciding with G(0)/G(1) arrest, and its attenuation in differentiated cells, these results suggest that the transient induction of DMBT1 is apparently specific at an early stage of gastric epithelial differentiation-like process, when it may play a role in cell fate decision. Consistent with such a potential function, we detected frequent abnormalities of the DMBT1 expression in the specimens of human gastric adenocarcinoma.

Detection of environmental androgens: a novel method based on enzyme-linked immunosorbent assay of spiggin, the stickleback (Gasterosteus aculeatus) glue protein.

We report the development and validation of a novel in vivo biomarker test for waterborne androgens. During breeding, male sticklebacks (Gasterosteus aculeatus) manufacture a glue protein, spiggin, in their kidneys that they use to build their nests. Spiggin production is under the control of androgens. Until now, however, it has only been possible to quantify its production by measurement of the height of kidney epithelial cells. In the present study, we report the development of an enzyme-linked immunosorbent assay (ELISA) for spiggin and demonstrate its application to the measurement of spiggin in the kidneys of female sticklebacks that have been exposed to androgens in water. Results from the ELISA procedure revealed a strong correlation with measurement of kidney epithelial cell height (r2 = 0.93). However, the ELISA was much quicker and had a considerably higher response range (100,000-fold vs fourfold). Clear, graded responses in spiggin production were obtained by exposing intact females to increasing concentrations of 17a-methyltestosterone and 5alpha-dihydrotestosterone over three-week test periods. The lowest effective concentrations for these two steroids were 100 ng/L and 3 micorg/L, respectively. Female sticklebacks that were exposed to pulp mill effluent also produced spiggin in their kidneys. Possession of an androgen-regulated protein by the female stickleback makes it a unique bioassay organism for detecting androgenic contamination in the aquatic environment.

Chemo-enzymatic synthesis of the glycosylated alpha-mating factor of Saccharomyces cerevisiae and analysis of its biological activity.

The effect of glycosylation on a bioactive peptide was studied using yeast Saccharomyces cerevisiae alpha-mating factor, which is composed of 13 amino acids. In this study, we prepared glycosylated alpha-mating factor by chemo-enzymatic synthesis. At first, N-acetylglucosaminyl alpha-mating factor (Trp-His-Trp-Leu-Gln(GlcNAc)-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr) was chemically synthesized by the solid-phase method. Then, using the transglycosylation activity of Mucor hiemalis endo-beta-N-acetylglucosaminidase, we synthesized glycosylated alpha-mating factor with a glutamine-linked sialo complex type oligosaccharide. The biological activity of alpha-mating factor derivatives was examined by means of a growth arrest assay using secreted-protease-defective a cells of S. cerevisiae. The results showed that the bioactivity of glycosylated alpha-mating factor was lower than that of native alpha-mating factor. However, when sialic acid was removed from the complex type sugar chain of glycosylated alpha-mating factor, its bioactivity was recovered. Glycosylated alpha-mating factor exhibited higher resistance against proteolysis than native alpha-mating factor. It was found that the bioactivity of N-acetylglucosaminyl alpha-mating factor was higher than that of alpha-mating factor. Circular dichroism studies indicated that a slight change in the structure of alpha-mating factor may influence its activity.

Transfusion support with RBCs from an Mk homozygote in a case of autoimmune hemolytic anemia following diphtheria-pertussis-tetanus vaccination.

BACKGROUND: Autoimmune hemolytic anemia (AIHA) in children, although unusual, is often associated with recent infection. Several reports have identified the diphtheria-pertussis-tetanus (DPT) vaccination as a possible trigger for AIHA. STUDY DESIGN AND METHODS: Life-threatening AIHA was diagnosed in a 6-week-old infant 5 days after receiving a DPT vaccination. The patient required daily transfusion and/or exchange transfusion for 3 weeks. RBCs from an Mk homozygote were found compatible with the patient's autoantibody. Transfusion of RBCs from an Mk homozygote and later RBCs from an individual (K.T.) with a variant glycophorin, Mi.VII, were required to sustain the patient's Hb level until autoantibody production ceased, as evidenced by a fall in antibody titer and the patient's Hct returning to normal. RESULTS: The DAT was positive (3+) with only anti-C3 on presentation. An IgM cold reactive autoantibody with probable anti-Pr specificity and high thermal amplitude (37 degrees C) was identified in the serum. The DAT was no longer positive after transfusion with compatible blood. CONCLUSION: This case represents life-threatening AIHA in an infant, temporally related to a DPT injection and responsive to a combination of immunosuppression and transfusion of rare compatible blood.

Transgenic B lymphocytes expressing a human cold agglutinin escape tolerance following experimental infection of mice by Mycoplasma pulmonis.

Several microbial infections, including Mycoplasma pneumoniae respiratory infection, are capable, in man, of transiently inducing the expression of anti-red blood cell autoantibody called cold agglutinins (CA). To analyze the mechanisms by which immune tolerance is broken following a mycoplasma infection, we used transgenic mice expressing a pathogenic human CA, designated CA-GAS, specific for sialylated carbohydrates. In these mice peripheral deletion of autoreactive B lymphocytes and receptor editing, prevent the development of autoimmune hemolytic anemia. Experimental infections of transgenic mice with Mycoplasma pulmonis resulted in a high anti-mycoplasma antibody response (despite a severe B cell depletion at the onset of infection), and an important induction of serum CA concentrations, reaching in some mice pathological titers. Whereas in naïve mice, only a small percentage of CA-expressing cells could be detected, in infected mice, a majority of circulating B lymphocytes were large B220(-) cells, which expressed the transgenic immunoglobulin. Immunization of the transgenic mice with keyhole limpet hemocyanin and Freund's adjuvant, to nonspecifically stimulate the expression of the passenger transgenes, only moderately increased the CA titers. These results indicate that M. pulmonis infection is capable of breaking immune tolerance in the CA-transgenic mice, in part through specific activation of CA-expressing B lymphocytes. This experimental infection mimics the induction of CA in humans and provide an animal model for studying the genesis of the autoimmune hemolytic anemia.

Paracrine effects of a uterine agglutinin are mediated via the sialic acids present in the rat uterine endometrium.

A 32 kDa estrogen-induced, sialic acid-specific agglutinin (P-SAS) was isolated from rat endometrium in its proestrus stage. To investigate the functional importance of P-SAS in the uterine milieu, specific binding assays were carried out with 125I-labeled P-SAS and different cellular components of the uterus (epithelial, stromal and myometrial cells), that were isolated from different stages of the estrus cycle. The results indicate that although the protein is secreted from the epithelial cells in the estrogenic phase, it binds specifically to the stromal cells, especially to those isolated from the diestrus stage of the estrus cycle. The specific binding, however, is seen to decrease with the progression of pregnancy. Scatchard analysis performed with varying amounts of 125I-P-SAS in the presence of excess cold P-SAS revealed that the binding occurs with a Ka = 1.69 x 10(8) M(-1). As P-SAS binds specifically to sialic acids on the stromal cell surface, further characterization of the sialic acid molecule to which P-SAS binds was carried out by gas liquid chromatography (GLC). The studies revealed that P-SAS preferentially binds to N-glycolylneuraminic acid, which is attached to the penultimate sugar of the stromal cell surface glycoprotein chain via alpha2,6 linkage. As P-SAS is further known to be mitogenic, the effect of P-SAS on cultured stromal cells was studied in vitro. The growth regulatory assays revealed that P-SAS induced 3H-thymidine uptake by stromal cells in culture. Thus, from the above observations, paracrine effects of P-SAS on the stromal cells and on the subsequent growth and development of the uterus can be assumed.

Ascites produced in rats without tubercle bacilli or tumor cells.

Intraperitoneal injection of rats with two doses of pertussis vaccine produces a small amount of ascitic fluid. Much larger amounts of fluid are produced when two spaced injections of the vaccine are preceded by a small amount of liquid petrolatum. A similar result is obtained by a single injection of pertussis vaccine emulsified in liquid petrolatum and Arlacel A. Ascites produced without tubercle bacilli or tumor cells may increase the use of rats for antibody production.

Inverse correlation between IgG-antihinge region and antierythrocyte autoantibody in chronic benign and malignant cold agglutination.

Previous reports provided evidence of an immunosuppressive role of natural anti-F(ab')2 antibodies. If suppressive anti-F(ab')2 antibodies also regulated the autoantibody production in cold agglutination, one would expect high titers of anti-F(ab')2 to be associated with low titers of cold agglutinins. Indeed, our previous studies revealed an inverse correlation between IgG-anti-F(ab')2 and cold agglutinins. Many previous experiments focused on anti-F(ab')2 of an antiidiotypic nature. Recent epitope mapping showed that anti-F(ab')2 of healthy persons is not an antiidiotype but recognizes a hinge region sequence. We attempted to answer the question whether this IgG-antihinge antibody is responsible for the previously described association between anti-F(ab')2 and cold agglutinins. IgG-antihinge and IgG-anti-F(ab')2 antibody was determined and statistically analyzed in the serum of 334 patients with cold agglutination. Our experiments revealed a strong correlation between the concentrations of antihinge and the previously described anti-F(ab')2 antibody. The anti-F(ab')2 activity was competitively inhibited by a synthetic hinge peptide. Moreover, patients with high antihinge titers had low cold agglutinin titers, and vice versa. A stratification according to cold agglutinin specificity and disease etiology showed that the inverse correlation is present only in anti-I and anti-i patients suffering from monoclonal B-lymphocyte proliferation. In conclusion, our results confirm the correlation previously described for anti-F(ab')2 antibody and antierythrocyte autoantibody and define for the first time an association between an idiotype-independent anti-IgG autoantibody and cold agglutinin.

The effect of anti-prolactin antibody on in vitro synthesis of a pregnancy-associated rat uterine glycoprotein.

Experiments were conducted to study the hormonal (estrogen [E2], progesterone [P4] and prolactin [PRL]), regulation of synthesis of a pregnancy-associated glycoprotein (named Uterine Agglutinin or UA) in the Day 4 post coital (p.c.) rat uterus with antibody administration and immunohistochemistry. Of the antibodies used, it was shown that anti-PRL antibody was the most effective in reducing in vitro UA synthesis. The results suggested that in vitro UA synthesis could be correlated to serum PRL levels as analyzed by radio-immunoassay. Binding studies revealed that PRL bound specifically to the stromal cells of the rat uterus where UA is produced and localized.

Experimental immunization of hamsters with an EDTA extract of Leptospira interrogans, serovar icterohaemorrhagiae.

The EDTA extract was prepared from the Leptospira interrogans serovar icterohaemorrhagiae. When inoculated subcutaneously in hamsters it conferred protection from challenge with virulent leptospires with a low dose (10 micrograms/ml) and low agglutinating antibody (40). The protection of animals obtained by the EDTA extract opens up the perspective of its use as a component of a vaccine for the control of leptospirosis.

[Significance and course of a cold agglutinin in Eperythrozoon suis infection of swine]. / Bedeutung und Verlauf eines Kälteagglutinins bei der Eperythrozoon suis-Infektion des Schweines.

After infection with Eperythrozoon suis, pigs began to produce cold agglutinins of immunoglobulin type IgM, that because of the similarity between the pathogenic antigen and antigen on the erythrocyte membrane caused agglomeration. The progression of this cold agglutinin was measured with an enzyme immunoassay especially invented for this purpose, and compared with serum-IgM, agglutination strength, pathogenic effect as well as number of erythrocytes in the blood. The cold agglutinin extracted from the erythrocyte membrane at 40 degrees C showed, in comparison to the initial figures, a higher level (1259 micrograms/ml) at 6 days after the illness peak, reaching its maximum (2435 micrograms/ml) at 12 days. Similar results were achieved at lower extraction temperatures (22 degrees C, 0 degree C). A high correlation could be shown between the levels of IgM and of cold agglutinin, as well as the parallel increase in the agglutination strength of the blood. At the time of maximal pathogenic effect, no agglutination of blood was observed. The number of erythrocytes decreased in acute phases of an attack to a constant mean of 2.32 Mill./microliters of blood and then increased, at the same rate as the cold agglutinin level decreased, almost reaching normal values. These experiments confirm the fact that an organism following an infection with Eperythrozoon suis, begins to produce cold agglutinins. Due to structural similarities between the pathogen and the erythrocyte antigen the cold agglutinin causes temperature dependent agglutination.(ABSTRACT TRUNCATED AT 250 WORDS)

Two acquired immunodeficiency syndrome-associated Burkitt's lymphomas produce specific anti-i IgM cold agglutinins using somatically mutated VH4-21 segments.

We analyzed the reactivity and the structure of the VH and VL segments of two IgM monoclonal antibodies (MoAbs) produced by spontaneously in vitro outgrowing cell lines, HBL-2 and HBL-3, established from two acquired immunodeficiency syndrome (AIDS) patients with Epstein-Barr virus (EBV)-negative Burkitt's lymphoma (BL). These B-cell clones were representative of the respective neoplastic parental clones, as determined by immunophenotypic and molecular genetic analysis. The IgM MoAbs were highly specific for the i determinant on red blood cells (cold agglutinins), but bound none of the other eight self and nine foreign antigens (Ags) tested, including those most commonly recognized by natural antibodies or autoantibodies. Structural analysis showed that the IgM MoAb VH segment sequences were 93.5% and 84.2% identical with that of the germline VH4-21 gene, which encodes the vast majority of cold agglutinins that are specific for the i/l carbohydrate Ag and are produced under chronic lymphoproliferative conditions. The HBL-2 MoAb VH4-21 gene segment was juxtaposed with 20P3 and JH6 genes and paired with a V lambda 1 segment, the sequence of which was 95.5% identical to that of the germline Humlv117 gene; the HBL-3 MoAb VH4-21 gene segment was juxtaposed with DXP'1 and JH5 genes and paired with a V lambda 1 segment, the sequence of which was 86.7% identical to that of the germline Humlv1L1 gene. The high degree of conservation of the VH4-21 gene in the human population, the nature of the nucleotide differences in the expressed VH4-21 segments, and the presence of nucleotide substitutions in the HBL-2 and HBL-3 IgM MoAb JH and/or J lambda segments suggested that the MoAb V segments underwent a process of somatic hypermutation. This was formally shown in the HBL-3 MoAb VH segment, by differentially targeted polymerase chain reaction amplification of the HBL-3 MoAb-producing cell genomic DNA. In addition, cloning and sequencing of the genomic DNA from fibroblasts of the same patient whose neoplastic B cells gave rise to the HBL-3 cell line yielded a germline copy of the VH4-21 gene. Thus, the expression of VH4-21 gene products may be involved in a self Ag-driven process of clonal B-cell expansion and selection associated with BL in these AIDS patients.

Antigenic competition in a multivalent foot rot vaccine.

The antigenic competition that occurs when pilus antigens of different serogroups are combined in multivalent vaccines for foot rot has been investigated using recombinant pilus antigens. Our prototype vaccine contains pili from nine serogroups of Dichelobacter nodosus which are expressed in Pseudomonas aeruginosa. Sheep inoculated with this multivalent vaccine were not as well protected against foot rot as those given the monovalent vaccine. Levels of agglutinating and total antibody specific for any particular pili serogroup were found to be significantly reduced in sheep vaccinated with six or more closely related pili. This effect was more pronounced for agglutinating antibody, which is thought to mediate protection, but was also observed with total antibody levels measured by ELISA. The antigenic competition was not associated with the total antigen load as a tenfold higher dose of monovalent pili induced high titres of antibody. Furthermore, distributing the vaccine to four sites, each draining to a different lymph node, failed to overcome the competition. Experiments with mixtures of monospecific sera indicate that the phenomenon is unlikely to be due to blocking of serogroup-specific protective antibodies by an excess of cross-reactive non-protective antibody elicited by heterologous pili.