Functional expression of human and mouse low density lipoprotein receptors in hybridomas

Though a mouse.human-human heterohybridoma, N12-16.63, secreting an antitetanus toxoid human monoclonal antibody grew well in a serum-free medium, its high producing subclone N12-69 required SSGF-I, a low density lipoprotein (LDL) from swine serum, or human-LDL (h-LDL) for growth. The growth-promoting action of SSGF-I was caused by its lipid fraction, and SSGF-I could be replaced completely with cholesterol in the presence of bovine serum albumin (BSA). Thus, cell line N12-69 is a cholesterol auxotroph of the heterohybridoma. N12-69 cells express both mouse and human LDL receptors on the cell surface in a ratio of 1:4. SSGF-I bound to both receptors with the same binding affinity, and h-LDL was also take up by the same receptors, though the affinity constant of the receptors for SSGF-I was 1.5 times stronger than that for h-LDL. The growth of N12-69 cells was completely inhibited by the addition of dextran sulfate, which is known to inhibit the binding of LDL to LDL receptors, to an SSGF-I or h-LDL containing medium but was not inhibited at all when dextran sulfate was added to a serum-free medium supplemented with cholesterol and BSA. Furthermore, an anti-human LDL receptor monoclonal antibody partially inhibited the growth of N12-69 cells in an SSGF-I or h-LDL containing medium. These findings suggest that N12-69 cells express both biologically active mouse and human LDL receptors on their cell surfaces and that SSGF-I or h-LDL is taken up by the both receptors to be utilized as a cholesterol source for the growth.     

Bovine monoclonal alloantibodies to blood group antigens prepared by murine x bovine or (murine x bovine) x bovine interspecies fusions.

In order to obtain monoclonal alloantibodies against bovine blood group antigens, lymph node cells from calves immunized with bovine red blood cells (RBC) were fused with either murine NSO/1 myeloma cells or a HAT sensitive murine x bovine heterohybridoma cell line. Both fusion partners resulted in heterohybridoma cell lines, producing monoclonal alloantibodies against bovine red blood cell antigens. Several clones produced antibodies against identical antigens and some of these clones have been further analysed. The antibodies produced by these selected cell lines have been compared with conventional polyclonal antisera used in bovine blood typing service. Thus extensive tests--including the ISAG Comparison Tests 1989/90 and 1991/92--have proved that monoclonal alloantibodies specific for the internationally recognized bovine red cell antigens A2, I1, O1, Q, A', B', Q', C1, R1, X1, S and Z have been produced. The Q, A', B', and C1 antibodies react weakly with certain phenogroups, whereas the A2, I1, O1, Q', R1, X1, S and Z antibodies have proved to be excellent blood typing reagents and have now substituted the polyclonal antisera in routine bovine blood typing in our laboratory.     

Contractile activity is required for the expression of neonatal myosin heavy chain in embryonic chick pectoral muscle cultures

The expression of neonatal myosin heavy chain (MHC) was examined in developing embryonic chicken muscle cultures using a monoclonal antibody (2E9) that has been shown to be specific for that isoform (Bandman, E., 1985, Science (Wash. DC), 227: 780-782). After 1 wk in vitro some myotubes could be stained with the antibody, and the number of cells that reacted with 2E9 increased with time in culture. All myotubes always stained with a second monoclonal antibody that reacted with all MHC isoforms (AG19) or with a third monoclonal antibody that reacted with the embryonic but not the neonatal MHC (EB165). Quantitation by ELISA of an extract from 2-wk cultures demonstrated that the neonatal MHC represented between 10 and 15% of the total myosin. The appearance of the neonatal isoform was inhibited by switching young cultures to medium with a higher [K+] which has been shown to block spontaneous contractions of myotubes in culture. Furthermore, if mature cultures that reacted with the neonatal antibody were placed into high [K+] medium, neonatal MHC disappeared from virtually all myotubes within 3 d. The effect of high [K+] medium was reversible. When cultures maintained in high [K+] medium for 2 wk were placed in standard medium, which permitted the resumption of contractile activity, within 24 h cells began to react with the neonatal specific antibody, and by 72 h many myotubes were strongly positive. Since similar results were also obtained by inhibiting spontaneous contractions with tetrodotoxin, we suggest that the development of contractile activity is not only associated with the maturation of myotubes in culture, but may also be the signal that induces the expression of the neonatal MHC.     

From insulin and insulin-like activity to the insulin superfamily of growth-promoting peptides: a 20th-century odyssey

In 1941, Gellhorn reported that administration of human blood to hypophysectomized/adrenodemedullated rats caused a fall in blood sugar. This was among the early demonstrations that human blood possesses glucose-lowering or insulin-like activity (ILA). Gellhorn assumed he had detected only insulin. During the 1960s, however, it became evident that plasma ILA contained at least two components: one, suppressible ILA (SILA), was inactivated by anti-insulin antibody and was therefore considered to be indistinguishable from pancreatic insulin; the other, nonsuppressible ILA (NSILA), was unaffected by anti-insulin antibody. Subsequent work resolved NSILA into insulin-like growth factors I and II (IGF-I and IGF-II), two 7.5 kilodalton peptides with potent mitogenic properties; established their identity with the somatomedins; and investigated both their therapeutic potential and role in the pathogenesis of neoplastic and other human diseases. Insulin and the IGFs exhibit striking homologies in amino acid composition and some degree of overlap in their signaling pathways and actions. Moreover, insulin-like proteins have been identified not only in all vertebrate classes but also in molluscs, insects, and worms. These observations are the basis for the hypothesis that the genes encoding vertebrate insulins and IGFs and invertebrate insulin-like molecules evolved from a common ancestral gene, and for the concept of an insulin superfamily of growth-promoting peptides.     

Identification of myosin heavy chain isoforms in skeletal muscle of four Southern African wild ruminants

The aim was to separate and characterize the myosin heavy chain (MHC) isoforms of four southern African wild ruminants, namely Blesbuck (Damaliscus dorcas phillipsi), Kudu (Tragelaphus strepsiceros), Black Wildebeest (Connochaetes gnou) and Blue Wildebeest (Connochaetes taurinus). Longissimus dorsi muscle samples were subjected to SDS-PAGE and Western blot analyses using antibodies raised against MHC isoforms. The specificity of these antibodies was assessed using immunohistochemistry combined with ATPase histochemistry, Three MHC isoforms were separated and the bands were identified from fastest to slowest migrating as MHC I, MHC IIx and MHC IIa. The mobility of the MHC isoforms was similar for all four species, including that of bovine, but differed from human muscle. Kudu muscle exhibited the lowest proportion of MHC I and the highest proportion of MHC IIx, whereas Blesbuck muscle had the least MHC IIx. The two Wildebeest species were intermediate in isoform content. In conclusion, when new species are studied, existing electrophoretic protocols may need to be modified to achieve quantifiable separation and isoform migration pattern must be verified in order to reach correct interpretations. Furthermore, antibody specificity may differ between techniques as well as species and needs confirmation.     

Production of rat monoclonal antibody from rat x mouse hybridoma cell lines using microencapsulation technology

Summary The cell growth and monoclonal antibody production characteristics of two rat x mouse heterohybridoma cell lines, designated 187.1 and M1/9.3, were investigated using a biocompatible microencapsulation technology. Both cell lines, derived from the fusion of immunized rat spleen cells with either the NS1 or X63Ag8.653 myeloma cell lines, were found to reach a maximum intracapsular cell density of 1.3 to 1.5×10⁷ cells/ml during a 27-d culture period. During this period, rat monoclonal antibody accumulated in the intracapsular space of both cultures to a final concentration of 2.0 to 2.8 mg/ml. Comparison of the concentration of rat monoclonal antibody in the extracapsular vs. the intracapsular space of both cultures indicated that significantly less than 1% of the antibody produced by the encapsulated hybridoma cells was capable of transiting the microcapsule membrane during the culture period. Due to the partition of the rat monoclonal antibody within the intracapsular space, the initial purity of the antibody harvested from 21-d microcapsule cultures of 187.1 and M1/9.3 cells was approximately 48 and 75% by weight, respectively. Analysis of the intracapsular protein by sodium dodecyl sulfoxide gel electrophoresis at different times during the culture period demonstrated that the principal contaminant associated with the unpurified antibody was bovine serum albumin.     

Interaction of indolicidin, a 13-residue peptide rich in tryptophan and proline and its analogues with model membranes

Indolicidin is a 13-residue broad-spectrum antibacterial peptide isolated from bovine neutrophils. The primary structure of the peptide ILPWKWPWWPWRR-amide (IL) reveals an unusually high percentage of tryptophan residues. IL and its analogues where proline residues have been replaced by alanine (ILA) and trp replaced by phe (ILF) show comparable antibacterial activitieso While IL and ILA are haemolytic, ILF does not have this property. Since aromatic residues would strongly favour partitioning of the peptide into the lipid bilayer interface, the biological activities of IL and its analogues could conceivably arise due perturbation of the lipid bilayer of membranes. We have therefore investigated the interaction of IL and its analogues with lipid vesicles. Peptides IL and ILA bind to lipid vesicles composed of phosphatidylcholine and phosphatidylethanol amine: phosphatidyl glycerol: cardiolipin. The position of λ_max and I^- quenching experiments suggest that the trp residues are localized at the membrane interface and not associated with the hydrophobic core of the lipid bilayer in both the peptides. Hence, membrane permeabilization is likely to occur due to deformation of the membrane surface rather than formation of transmembrane channels by indolicidin and its analogues. Peptides ILA, IL and ILF cause the release of entrapped carboxyfluorescein from phosphatidyl choline vesicles. The peptide-lipid ratios indicate that ILF is less effective than IL and ILA in permeabilizing lipid vesicles, correlating with their haemolytic activities. An erratum to this article is available at .     

Detection of antibodies in serum and cerebrospinal fluid to Toxoplasma gondii by indirect latex agglutination test and enzyme-linked immunosorbent assay.

Sensitivity of anti-Toxoplasma antibody (IgG) test by enzyme-linked immunosorbent assay (ELISA) was evaluated in comparison with indirect latex agglutination (ILA) using 2,016 paired human samples of serum and cerebrospinal fluid (CSF). The samples were collected from neurologic patients in Korea with mass lesions in central nervous system (CNS) as revealed by imaging diagnosis (CT/MRI). When the sera were screened for anti-Toxoplasma antibody by ILA, 76 cases(3.8%) were positive (1:32 or higher titers). In the paired samples of CSF, no positive reactions were observed. When ELISA was performed using PBS extract of Percoll purified tachyzoites as antigen, cut-off absorbance was determined as 0.40 for serum and 0.27 for CSF tests. The antibody positive rates by ELISA were 7.0% in serum and 5.6% in CSF. Of them, 40 cases (2.0%) showed positive reactions in both serum and CSF. The antibody positive rates were higher in groups older than 40 years. The rates were higher in male (4.7% by ILA, 8.3% by ELISA) than in female (2.2% by ILA, 5.0% by ELISA). The rates in CSF showed no such sex difference. ELISA showed twice higher positive rates when serum was tested, and was sensitive enough to detect specific antibodies in CSF. Etiologic relations between positive antibody tests and CNS lesions remained unknown.     

Deletion of one of the duplicated Hsp70 genes causes hereditary myopathy of diaphragmatic muscles in Holstein-Friesian cattle

Heat-shock protein 70 (Hsp70) is a major chaperone that folds protein and prevents aggregation. The Hsp70 family contains both constitutive and stress-inducible forms. In humans, two of the inducible Hsp70 genes are located within the human major histocompatibility complex (MHC) on 6p21.3, as a duplicated locus, 12 kb apart from each other. We report that loss of one of the duplicated Hsp70 genes, the bovine homologue within the bovine MHC, is responsible for hereditary myopathy of diaphragmatic muscles (HMDM) in Holstein-Friesian cattle. Although the remaining Hsp70 gene is intact, Hsp70 protein levels are dramatically decreased in affected cattle. In normal diaphragmatic muscle, Hsp70 binds several proteins involved in energy metabolism including glycogen phosphorylase (PYGM). Immunohistochemical staining indicated that PYGM accumulated in the HMDM-specific core-like structures in affected cattle. Misfolding of energy-related proteins due to Hsp70 deficiency might lead to protein aggregation and muscle fibre degeneration.     

Increased leaf angle1, a Raf-like MAPKKK that interacts with a nuclear protein family, regulates mechanical tissue formation in the Lamina joint of rice

Mitogen-activated protein kinase kinase kinases (MAPKKKs), which function at the top level of mitogen-activated protein kinase cascades, are clustered into three groups. However, no Group C Raf-like MAPKKKs have yet been functionally identified. We report here the characterization of a rice (Oryza sativa) mutant, increased leaf angle1 (ila1), resulting from a T-DNA insertion in a Group C MAPKKK gene. The increased leaf angle in ila1 is caused by abnormal vascular bundle formation and cell wall composition in the leaf lamina joint, as distinct from the mechanism observed in brassinosteroid-related mutants. Phosphorylation assays revealed that ILA1 is a functional kinase with Ser/Thr kinase activity. ILA1 is predominantly resident in the nucleus and expressed in the vascular bundles of leaf lamina joints. Yeast two-hybrid screening identified six closely related ILA1 interacting proteins (IIPs) of unknown function. Using representative IIPs, the interaction of ILA1 and IIPs was confirmed in vivo. IIPs were localized in the nucleus and showed transactivation activity. Furthermore, ILA1 could phosphorylate IIP4, indicating that IIPs may be the downstream substrates of ILA1. Microarray analyses of leaf lamina joints provided additional evidence for alterations in mechanical strength in ila1. ILA1 is thus a key factor regulating mechanical tissue formation at the leaf lamina joint.     

Proteolytic conversion of insulin-like growth factors to an acidic form(s).

The relative amounts of the various forms of bioassayable insulin-like growth factors (IGF) isolated from human serum or serum fraction Cohn IV-1 depend on the purification procedure. With acid gel filtration or acid/ethanol extraction as the initial step, IGF-II (pI approximately 6.5) was the most abundant (40-70%) followed by somatomedin A (pI approximately 7.4; 15-23%), an acidic form of insulin-like activity (ILA pI 4.8) (13-21%) and IGF-I (pI approximately 8.5; 5-27%). If, however, pH 5.5 ion-exchange chromatography on SP-Sephadex was used prior to acid gel filtration, the acidic pI 4.8 form was the major (greater than 90%) species recovered and was accompanied by a quantitative loss of the other IGF species. This suggested a possible conversion of IGF-I, somatomedin A and/or IGF-II to the acidic ILA pI 4.8 form(s) during the SP-Sephadex procedure. Further experiments indicated that differences in the yields of ILA pI 4.8 were not due simply to differences in the initial pH conditions of the various methods (i.e. acid versus neutral), although exposure to pH 9.7 (a pH experienced during elution of IGF activity from the SP-Sephadex) did appear to play a role. The involvement of the carrier protein in the conversion process was tested by subjecting carrier-free IGF-I and IGF-II to the SP-Sephadex procedure. No conversion of the free forms to ILA pI 4.8 occurred. To examine the possible role of proteinase in the conversion of IGFs to ILA pI 4.8, SP-Sephadex chromatography was performed in the presence of a broad spectrum proteinase inhibitor. The IGF distribution pattern obtained closely resembled the 'normal' pattern seen with acid gel filtration, indicating that proteinase inactivation had prevented conversion to ILA pI 4.8. These data suggest that proteolytic conversion of IGF-I, somatomedin A and IGF-II to more acidic ILA pI 4.8 form(s) (i) occurs during SP-Sephadex chromatography, (ii) is not prevented simply by prior acid exposure, and (iii) takes place only when IGF-I and -II are in their high-Mr carrier-bound forms. Since IGF-I and IGF-II, although homologous, have unique amino acid sequences, the conversion of both IGFs implies that at least two acidic ILA forms exist. Nevertheless, because ILA pI 4.8 retains the full spectrum of IGF bioactivities in vitro, and significant quantities are present in normal human serum (21%), it would suggest that proteolytic conversion of IGF-I, somatomedin A and IGF-II to ILA pI 4.8 in vivo may be a physiologically significant event.     

Myosin isoforms and cell heterogeneity in vascular smooth muscle. I. Developing and adult bovine aorta

Monoclonal anti-smooth muscle (SM-E7, SM-F11, and BF-48) and anti-nonmuscle (NM-A9 and NM-G2) myosin antibodies, Western blotting, and immunocytochemical procedures were used to study myosin isoform composition and distribution in the smooth muscle (SM) cells of bovine aorta differentiating in vivo and in vitro. Two myosin heavy chain (MHC) isoforms were identified by SM-E7 in adult aorta: SM-MHC-1 (Mr = 205 kDa) and SM-MHC-2 (Mr = 200 kDa), respectively. When tested with the SM-F11 antibody, SM-MHC-2 isoform showed distinct antigenic properties compared to SM-MHC-1. Two bands of 205 and 200 kDa were also present in the aortic SM tissue from 3-month-old fetus and were equally recognized by the BF-48 antibody. The 200-kDa SM myosin isoform was labeled by SM-F11 but not by SM-E7, thus indicating the existence of a fetal-specific SM-MHC-2 isoform. At the cellular level, both developing and adult bovine aortic tissues showed the existence of distinct patterns of myosin isoform expression. Three or even more aortic cell populations are differently distributed in areas which appear as (1) a network of interconnecting sheet-like or compact tissue (early fetus) and (2) enriched of collagenous-elastic or muscular tissue (adult animal). In addition, the SM-MHC-2 isoform of the fetal type appears to be uniquely distributed in cultured SM cells grown in vitro from adult bovine aortic explants. Our data indicate that in bovine aorta (1) MHC isoform expression is developmentally regulated and (2) the distribution of myosin isoforms is heterogenous both among and within aortic cells. These findings may be related to the distinct physiological properties displayed by SM during vascular myogenesis.     

Retroviral antibody binding of the MHC class II molecule: a biochemical influence on CD4 T cell differentiation in HIV infection?

Retroviral antibody capable of binding to the major histocompatibility complex (MHC) Class II molecule has been documented in human immunodeficiency virus-1 (HIV-1)-infected patients. Interactions between the MHC Class II receptor and the T-cell receptor (TCR) are central to the immune response. Importantly, retroviral antibody possesses a much higher binding affinity for the MHC Class II receptor, when compared to the TCR. Experiments have manipulated a number of factors related to antigen-presenting cell (APC) interaction with differentiating T-cells. These studies have observed the effects of lowering antigen dose and reducing ligand density on precursor Th (T helper) cell differentiation. Studies have also examined the effect of downregulated MHC Class II receptors and co-stimulatory molecules on APC-Th cell interaction. In addition, the sequestration of antigens away from the Class II processing pathway has been studied. These investigations reveal a general trend that can determine whether a naive CD4 T-cell becomes a Th1 or Th2-like cell. If the experimental manipulation weakens the APC-Th cell interaction, a weak ligating TCR signal results. Consequently, a weak ligating TCR signal can influence precursor Th cells to become Th2-like cells. Retroviral antibody binding of MHC Class II receptors may mimic a number of experimental conditions responsible for creating a weak ligating TCR signal.     

Molecular and cellular studies of tryptophanyl-tRNA synthetases using monoclonal antibodies. Remarkable variations in the content of tryptophanyl-tRNA synthetase in the pancreas of different mammals

The content of Trp-tRNA synthetase in pancreas and liver of cattle, sheep, swine, rat, rabbit and man was assayed by direct radioimmunoblotting with a 125I-labelled monoclonal antibody Am1, specifically interacting with any eukaryotic Trp-tRNA synthetase. Its content in the organs studied, with the exception of bovine and sheep pancreas, was found to be 0.002-0.012% of total proteins. The enzyme content in bovine pancreas was about 0.2% of total proteins, i.e. 70 times higher than in bovine liver; similar correlations were found for sheep. The Trp-tRNA synthetase levels in each organ varied from animal to animal of the same species by not more than a factor of four; these individual variations cannot affect the conclusion about the profound differences in the levels of the enzyme in pancreases of Ruminantia and of the other mammalians. As shown by indirect immunofluorescence technique, bovine Trp-tRNA synthetase is mainly located in the exocrine part of the pancreas. Moreover, the immunoreactive material is detectable also in bovine (not human) pancreatic juice. The abnormally high Trp-tRNA synthetase content in the ruminant pancreas may be connected with unknown function(s) of this protein somehow related to the peculiarities of digestion of these mammals.     

Comparative analysis of the bovine MHC class IIb sequence identifies inversion breakpoints and three unexpected genes

The bovine major histocompatibility complex (MHC) or BoLA is organized differently from typical mammalian MHCs in that a large portion of the class II region, called class IIb, has been transposed to a position near the centromere on bovine chromosome 23. Gene mapping indicated that the rearrangement resulted from a single inversion, but the boundaries and gene content of the inverted segment have not been fully determined. Here, we report the genomic sequence of BoLA IIb. Comparative sequence analysis with the human MHC revealed that the proximal inversion breakpoint occurred approximately 2.5 kb from the 3' end of the glutamate-cysteine ligase, catalytic subunit (GCLC) locus and that the distal breakpoint occurred about 2 kb from the 5' end from a divergent class IIDRbeta-like sequence designated DSB. Gene content, order and orientation of BoLA IIb are consistent with the single inversion hypothesis when compared with the corresponding region of the human class II MHC (HLA class II). Differences with HLA include the presence of a single histone H2B gene located between the proteasome subunit, beta type, 9 (PSMB9) and DMB loci and a duplicated TAP2 with a variant splice site. BoLA IIb spans approximately 450 kb DNA, with 20 apparently intact genes and no obvious pseudogenes. The region contains 227 simple sequence repeats (SSRs) and approximately 167 kb of retroviral-related repetitive DNA. Nineteen of the 20 genes identified in silico are supported by bovine EST data indicating that the functional gene content of BoLA IIb has not been diminished because it has been transposed from the remainder of BoLA genes.     

Variation in T cell responses to ovalbumin in cattle: evidence for Ir gene control

Genetic control of immune responsiveness in cattle was investigated using an antigen-dependent T cell proliferation assay in vitro. Bovine T cell proliferative responses to ovalbumin were dependent upon major histocompatibility complex (MHC) class II molecules. Responses of an unrelated panel of animals to a limiting concentration of ovalbumin after a single immunization were compared. Two discrete patterns of response were observed. One group of animals had low or non-responses which were not significantly different from the preimmune levels. Another group of animals showed significant responses. After a second immunization the majority of low responders remained low responders. There was no significant correlation between bovine MHC class I BoLA haplotype and magnitude of response within this group of unrelated animals. However, the magnitude of the T cell responses by two half-sib family groups segregated with BoLA haplotypes inherited from the sire. In contrast no significant correlation with antibody responses in vivo could be demonstrated. We suggest that the observed variation in T cell response is linked to bovine MHC class II immune response (Ir) genes.     

A defect in mobilization of cholesteryl esters in rabbit macrophages

Macrophages provide an important way for cholesteryl esters to accumulate in tissues in pathologic amounts. We studied cholesteryl ester metabolism in thioglycollate-induced peritoneal macrophages obtained from normocholesterolemic and hypercholesterolemic rabbits. The macrophage preparations from normocholesterolemic rabbit (MN cells) had 26 nmol esterified cholesterol/mg cellular protein, incorporated 1 nmol of labeled oleate into cholesteryloleate/2 h per mg cellular protein and had an acyl-coenzyme A:cholesterol acyltransferase activity of 22 pmol cholesterylpalmitate formed/min per mg protein in isolated membranes. The macrophage preparations from hypercholesterolemic rabbits (MHC cells) contained a 12-fold greater mass of cholesteryl ester, had an 8-times higher rate of formation of cholesteryloleate, and had 3-times more acyl-coenzyme A:cholesterol acyltransferase activity in the isolated membranes. When a cholesterol acceptor (10% fetal bovine serum or 10 mg of lipid-free fetal bovine serum protein) was added to the culture medium of rabbit MHC cells, the MHC cells retained more than 70% of their cholesteryl esters after 48 h of incubation. In contrast, when a cholesterol acceptor (10% fetal bovine serum) was added to the medium of thioglycollate-induced, cholesterol-enriched macrophages from mice, the mice macrophages retained only 19% of their cholesteryl esters after 48 h of incubation. The limited capacity of rabbit macrophages to release unesterified cholesterol from stored cytoplasmic cholesteryl esters to an exogenous acceptor may be related to the propensity of rabbits to develop atherosclerotic lesions.     

Immunoreactive insulin and insulin-like activity of the blood in ontogenesis of the hen]

Studies have been made on immunoreactive insulin (IRI) and insulin-like activity (ILA) in the blood serum of chick embryos (from the 10th day of incubation), chicks and adult hens up to 1 year old. It was shown that IRI content of embryonic blood is relatively low and remains approximately constant during incubation. During postnatal ontogenesis, the level of IRI increases, the increase being most significant at the 1st day after hatching and between the 2nd and the 5th months. With respect to IRI level, 5-month chicks are similar to adult hens. Being assayed by the method of isolated epididymal rat fat, ILA was not found in the blood serum of chick embryos. It was observed in all test samples only from the 30th day after hatching. It is suggested that at this period of postnatal life, some factors are formed in the blood which increase ILA without changes of the insulin content of the blood. After the 30th day, no evident shifts were observed in ILA, although it reached maximum in adult hens. By absolute values, ILA of the blood in chicks was several times higher than the corresponding levels of IRI.     

Alloantibodies against MHC class I: a novel mechanism of neonatal pancytopenia linked to vaccination

Fetal/neonatal alloimmune thrombocytopenia is a frequent disease in humans where alloantibodies against platelet Ags lead to platelet destruction and hemorrhage. Although a role in the disease for Abs against MHC has been suspected, this has not been formally demonstrated. Since 2007, a hemorrhagic syndrome due to thrombocytopenia and designated as bovine neonatal pancytopenia (BNP) has been recognized in calves in several European countries. An inactivated antiviral vaccine is strongly suspected to be involved in this syndrome because of its highly frequent use in the dams of affected calves. In this study, we show that BNP is an alloimmune disease, as we reproduced the signs by transferring serum Abs from vaccinated BNP dams into healthy neonatal calves. Ab specificity was strongly associated with the presence of allogeneic MHC class I Abs in the dams. MHC class I staining was also observed on Madin-Darby bovine kidney cells, a cell line related to the one used to produce the vaccine Ag. Our report emphatically demonstrates that alloimmunization against MHC class I is associated with a substantial risk of developing cytopenia-associated syndromes in neonates when a cell line of the same species is used to produce an inactivated vaccine injected into the mother.