Small cells in hepatoblastoma lack "oval" cell phenotype.

Hepatoblastoma, a childhood tumor of the liver, is composed of epithelial and mesenchymal elements in varying proportions and at various stages of differentiation. The epithelial element recapitulates the stages of hepatocyte development from the primitive blastema through embryonal hepatocytes to fetal hepatocytes. The blastemal or undifferentiated cells have been postulated to represent neoplastic hepatocyte progenitor cells. In this study, we examine the immunophenotype of the various epithelial cells of hepatoblastoma with special emphasis on the small undifferentiated cell component and compare it with that of adult hepatocytes and hepatic stem (oval) cells. Putative stem cells in the liver can express all of the following markers: alpha-feto protein, CK19 (OV-6), chromogranin A, Bcl-2, HepPar-1, and alpha1 microglobulin. The latter, like alpha-feto protein, is a plasma protein synthesized by hepatocytes. Both alpha1 microglobulin and HepPar-1 are expressed in fetal liver cells as early as 7 weeks of intrauterine life. They are also expressed in hepatocellular carcinoma and in hepatocytic cell lines derived from normal fetal or adult liver. Formalin-fixed, paraffin-embedded archival tissues from 10 predominantly epithelial hepatoblastomas were immunostained with antibodies directed against CD 34, alpha1 microglobulin, Bcl-2, HepPar 1, and CK19 using the avidin-biotin-peroxidase method. The undifferentiated small cell component did not express any of the markers studied, namely, Bcl-2, HepPar-1, alpha(1) microglobulin, CD34, or CK19. Hepatocyte-like cells were alpha1 microglobulin- and HepPar-1-positive, with the intensity of staining correlating with the degree of hepatocytic differentiation. Bcl-2 expression was restricted to areas of ductular differentiation. CK19 was detected in foci that showed duct formation. The small cells of hepatoblastoma did not express HepPar-1, Bcl-2, CK19, alpha1 microglobulin, or CD34, markers that characterize the immunophenotype of hepatic stem cells ("oval" cells). Thus, this observation raises the following questions: (1) is "hepatoblastoma" a misnomer? (2) is the expression of tumor antigens dysregulated in hepatoblastoma? (3) does the liver have two different types of progenitor cells, oval cells and blastemal cells, with differing immunophenotypes? and (4) do the blastemal cells, rather than oval cells, represent the more primitive progenitor cells of the liver?

Distribution of iodine 125-labeled alpha1-microglobulin in rats after intravenous injection.

The 28-kd plasma protein alpha(1)-microglobulin is found in the blood of mammals and fish in a free, monomeric form and as high-molecular-weight complexes with molecular masses above 200 kd. In this study, iodine 125-labeled free and high-molecular weight rat alpha(1)-microglobulin (a mixture of alpha(1)-microglobulin/alpha(1)-inhibitor-3 and alpha(1)-microglobulin/fibronectin complexes) were injected intravenously into rats. The distribution of the proteins was measured by using scintillation camera imaging. Both forms of (125)I-labeled alpha(1)-microglobulin were rapidly cleared from the blood, with a half-life of 2 and 16 minutes for the initial and late phase, respectively, for free alpha(1)-microglobulin; and a half-life of 3 and 130 minutes for the initial and late phase, respectively, for the complexes. After 45 minutes, 6%, 16%, 27%, 13%, and 34% of the free (125)I-labeled alpha(1)-microglobulin and 18%, 21%, 6%, 10%, and 42% of the (125)I-labeled alpha(1)-microglobulin complexes were found in the blood, gastrointestinal tract, kidneys, liver, and the remainder of the body, respectively. The local distribution of injected (125)I-labeled alpha(1)-microglobulin in intestines and kidneys was investigated by microscopy and autoradiography. In the intestine, both forms were distributed in the basal layers, villi, and luminal contents. The results also suggested intracellular labeling of epithelial cells. Well-defined local regions containing higher concentrations of injected protein could be seen in the intestine. In the kidneys, both forms were found mostly in the cortex. Free (125)I-labeled alpha(1)-microglobulin was found predominantly in epithelial cells of a subset of the tubules, whereas the (125)I-labeled complexes were more evenly distributed. Intracellular labeling was indicated for both alpha(1)-microglobulin forms. The results thus indicate a rapid transport of (125)I-labeled alpha(1)-microglobulin from the blood to most tissues.

alpha(1)-Microglobulin: a yellow-brown lipocalin.

alpha(1)-Microglobulin, also called protein HC, is a lipocalin with immunosuppressive properties. The protein has been found in a number of vertebrate species including frogs and fish. This review summarizes the present knowledge of its structure, biosynthesis, tissue distribution and immunoregulatory properties. alpha(1)-Microglobulin has a yellow-brown color and is size and charge heterogeneous. This is caused by an array of small chromophore prosthetic groups, attached to amino acid residues at the entrance of the lipocalin pocket. A gene in the lipocalin cluster encodes alpha(1)-microglobulin together with a Kunitz-type proteinase inhibitor, bikunin. The gene is translated into the alpha(1)-microglobulin-bikunin precursor, which is subsequently cleaved and the two proteins secreted to the blood separately. alpha(1)-Microglobulin is found in blood and in connective tissue in most organs. It is most abundant at interfaces between the cells of the body and the environment, such as in lungs, intestine, kidneys and placenta. alpha(1)-Microglobulin inhibits immunological functions of white blood cells in vitro, and its distribution is consistent with an anti-inflammatory and protective role in vivo.

Immunocalins: a lipocalin subfamily that modulates immune and inflammatory responses.

A subset of the lipocalins, notably alpha(1)-acid glycoprotein, alpha(1)-microglobulin, and glycodelin, exert significant immunomodulatory effects in vitro. Interestingly, all three are encoded from the q32-34 region of human chromosome 9, together with at least four other lipocalins (neutrophil gelatinase-associated lipocalin, complement factor gamma-subunit, tear prealbumin, and prostaglandin D synthase) that also may have anti-inflammatory and/or antimicrobial activity. This review addresses important features of this genetically linked subfamily of lipocalins (involvement with the acute phase response, immunomodulatory and anti-inflammatory properties, the tissue localization, complex formation with other proteins and receptors, etc.). It is likely that these proteins have evolved to be an integrated part of the body's defense system as part of the extended cytokine network. Its members exert a regulatory, dampening influence on the inflammatory cascade, thereby protecting against tissue damage from excessive inflammation. That most major mammalian allergens are lipocalins may reflect this connection of lipocalins with the immune system. We propose that this immunologically active lipocalin subset be named the 'immunocalins', signifying not only the structural homology and close genetic linkage of its members, but also their protective involvement with immunological and inflammatory processes. As immune mediators, immunocalins appear to use at least three interactive sites: the lipocalin 'pocket', binding sites for other plasma proteins, and binding sites for cell surface receptors.

Tissue distribution of the lipocalin alpha-1 microglobulin in the developing human fetus.

Alpha-1 microglobulin (alpha(1)m), a lipocalin, is an evolutionarily conserved immunomodulatory plasma protein. In all species studied, alpha(1)m is synthesized by hepatocytes and catabolized in the renal proximal tubular cells. alpha(1)m deficiency has not been reported in any species, suggesting that its absence is lethal and indicating an important physiological role for this protein To clarify its functional role, tissue distribution studies are crucial. Such studies in humans have been restricted largely to adult fresh/frozen tissue. Formalin-fixed, paraffin-embedded multi-organ block tissue from aborted fetuses (gestational age range 7-22 weeks) was immunohistochemically examined for alpha(1)m reactivity. Moderate to strong reactivity was seen at all ages in hepatocytes, renal proximal tubule cells, and a subset of pancreatic islet cells. Muscle (cardiac, skeletal, or smooth), adrenal cortex, a scattered subset of intestinal mucosal cells, tips of small intestinal villi, and Leydig cells showed weaker and/or variable levels of reactivity. Connective tissue stained with variable location and intensity. The following cells/sites were consistently negative: thymus, spleen, hematopoietic cells, lung parenchyma, glomeruli, exocrine pancreas, epidermis, cartilage/bone, ovary, seminiferous tubules, epididymis, thyroid, and parathyroid. The results underscore the dominant role of liver and kidney in fetal alpha(1)m metabolism and provide a framework for understanding the functional role of this immunoregulatory protein.

An antigen reacting with das-1 monoclonal antibody is ontogenically regulated in diverse organs including liver and indicates sharing of developmental mechanisms among cell lineages.

The monoclonal antibody designated mAb Das-1, which was generated against a colon epithelial protein, reacts with the normal biliary epithelium and keratinocytes, which are among targets of tissue injury in ulcerative colitis. Moreover, mAb Das-1 reacts with abnormal cells in Barrett's esophagus and chronic cystitis profunda, as well as so-called 'oval cells' in the adult liver, which are considered oncogenic progenitor cells. To establish ontogenic regulation of mAb Das-1 reactivity, we studied 7- to 24-week-old human fetuses by immunohistochemistry. In liver, mAb Das-1 reactivity was further correlated with glycogen, dipeptidyl peptidase IV, glucose-6-phosphatase and gamma-glutamyl transpeptidase expression. mAb Das-1 reacted with cells in organs arising from the pharyngeal cleft (thymus), primitive gut (oral cavity, pharynx, lung, esophagus, stomach, biliary tree, pancreas, liver, colon), ureteric bud (renal tubules, collecting duct), mesonephros (kidney, testis), mesoderm (muscle) and elsewhere (skin, adrenal cortex). In distinction from the adult liver, mAb Das-1 staining was more pronounced in hepatoblasts compared with biliary cells. In adult tissues, however, mAb Das-1 reactivity was restricted to the colon, biliary epithelium, keratinocytes, and ciliary body. These data indicated that the mAb Das-1 recognized epitopes in fetal cells of diverse ectodermal, mesodermal and endodermal origin, compatible with sharing of lineage mechanisms in tissues. Reactivation of mAb Das-1 staining in epithelial precancerous conditions, including carcinomas arising in these organs, is compatible with oncofetal regulation of the antigen, which will facilitate analysis of cell subpopulations during organ development, regeneration and oncogenesis.

Histologic distribution and biochemical properties of alpha 1-microglobulin in human placenta.

PROBLEM: The embryo is protected from immunologic rejection by the mother, possibly accomplished by immunosuppressive molecules located in the placenta. We investigated the distribution and biochemical properties in placenta of the immunosuppressive plasma protein alpha 1-microglobulin. METHOD OF STUDY: Placental alpha 1-microglobulin was investigated by immunohistochemistry and, after extraction, by electrophoresis, immunoblotting and radioimmunoassay. RESULTS: alpha 1-Microglobulin staining was observed in the intervillous fibrin and in syncytiotrophoblasts, especially at sites with syncytial injury. Strongly stained single cells in the intervillous spaces and variably stained intravillous histiocytes were noted. Solubilization of the placenta-matrix fraction and placenta membrane fraction released predominantly the free form of alpha 1-microglobulin, but, additionally, an apparently truncated form from the placenta-membrane fraction. The soluble fraction of placenta contained two novel alpha 1-microglobulin complexes. CONCLUSIONS: The biochemical analysis indicates the presence in placenta of alpha 1-microglobulin forms not found in blood. The histochemical analysis supports the possibility that alpha 1-microglobulin may function as a local immunoregulator in the placenta.

Effect of a conjugate of polymyxin B-dextran 70 in horses with experimentally induced endotoxemia.

OBJECTIVE: To determine the efficacy of polymyxin B-dextran 70 (PBD) for treatment of endotoxemic horses. ANIMALS: 15 horses during study 1 and 6 horses during study 2. PROCEDURES: 3 groups were used in study 1. Horses in groups 1 and 2 were given 30 ng of lipopolysaccharide (LPS)/kg of body weight, IV, over 60 minutes. Horses in group 3 were given saline (0.9% NaCl) solution. Beginning 15 minutes before LPS infusion and continuing for 75 minutes, horses in groups 1 and 3 were given PBD, IV. Horses in group 2 were given dextran 70. Blood samples were obtained for hemograms and determination of cytokine, lactate, and prostanoid concentrations. In study 2, horses were given ketoprofen (2.2 mg/kg) or saline solution 15 minutes before infusion of PBD. Fourteen days later, treatments were reversed, using a crossover design. Blood samples were obtained for measurement of thromboxane B2 (TXB2) concentration. RESULTS: For study 1, prior treatment with PBD completely blocked endotoxin-induced changes for heart and respiratory rates, rectal temperature, WBC count, and plasma tumor necrosis factor, interleukin 6, TXB2, and prostaglandin F1 concentrations. There was transient tachypnea, sweating, and increased plasma TXB2 concentration in horses given PBD (with or without LPS). Prior treatment with ketoprofen eliminated all PBD-induced signs and prevented the increase in plasma TXB2 concentration. CONCLUSIONS: Signs of endotoxemia were prevented in horses by treatment with PBD, although its use was associated with mild adverse effects. CLINICAL RELEVANCE: When used in combination with a cyclooxygenase-inhibiting drug, PBD has potential for treatment of horses with endotoxemia.

Therapeutic efficacy of a polymyxin B-dextran 70 conjugate in experimental model of endotoxemia.

Numerous studies have suggested that lipopolysaccharide (LPS), a major component of the cell wall of gram-negative bacteria, is responsible for the initiation of gram-negative septic shock. Previously, others have designed therapeutic regimens to target the biologically active lipid A region of LPS by either neutralization of the biological properties of LPS or enhancement of clearance of this molecule. One such compound capable of neutralizing lipid A is the antibiotic polymyxin B. However, the clinical utility of polymyxin B is limited by its toxicity. We therefore covalently conjugated this antibiotic to the high-molecular-weight polysaccharide dextran 70, resulting in reduced toxicity of polymyxin B but retention of its endotoxin-neutralizing ability. The studies described in this report were designed to test the in vivo efficacy of this compound in an experimental animal model of gram-negative septic shock. Mice were administered graded doses of Escherichia coli or Pseudomonas aeruginosa along with D-galactosamine and the antibiotic imipenem. We had previously determined that antibiotic chemotherapy provides significant protection against E. coli-mediated lethality with smaller doses of bacteria; however, the antibiotic does not provide protection against larger doses of bacteria, but it is effective at killing the bacterial inoculum in vivo. Administration of the polymyxin B-dextran 70 conjugate provided significant protection when given with an antibiotic but was not effective by itself. A requirement for a pretreatment period prior to E. coli challenge was shown to depend upon the bacterial challenge dose. In other studies using this D-galactosamine sensitization model, we demonstrated that the lipid A-specific conjugate had no effect on lethality caused by staphylococcus aureus or tumor necrosis factor alpha. The results of these studies indicate that this compound is effective in preventing lethal gram-negative septic shock in mice and may be useful as a potential therapeutic agent in humans as well.

Primate antibodies to components of the human immune system.

The feasibility to raise nonhuman primate antibodies against selected components of the human immune system was tested. The immunogens were whole cells (human T lymphocytes) or purified, recombinant human proteins (cytokines: TNF alpha or GM-CSF; soluble forms of cell surface antigens: sCD4 or sCD25). Significant immunizations, yielding functionally relevant antibodies, were readily achieved in rhesus monkeys, but, not surprisingly, may be less frequent in chimpanzees. The results suggest a general strategy for production of therapeutically useful MAB.

Purification of antibodies using protein L-binding framework structures in the light chain variable domain.

Protein L from the bacterial species Peptostreptococcus magnus binds specifically to the variable domain of Ig light chains, without interfering with the antigen-binding site. In this work a genetically engineered fragment of protein L, including four of the repeated Ig-binding repeat units, was employed for the purification of Ig from various sources. Thus, IgG, IgM, and IgA were purified from human and mouse serum in a single step using protein L-Sepharose affinity chromatography. Moreover, human and mouse monoclonal IgG, IgM, and IgA, and human IgG Fab fragments, as well as a mouse/human chimeric recombinant antibody, could be purified from cultures of hybridoma cells or antibody-producing bacterial cells, with protein L-Sepharose. This was also the case with a humanized mouse antibody, in which mouse hypervariable antigen-binding regions had been introduced into a protein L-binding kappa subtype III human IgG. These experiments demonstrate that it is possible to engineer antibodies and antibody fragments (Fab, Fv) with protein L-binding framework regions, which can then be utilized in a protein L-based purification protocol.

Characterization of monoclonal anti-alpha 1-microglobulin antibodies: binding strength, binding sites, and inhibition of lymphocyte stimulation.

Eleven monoclonal antibodies (MoAb) directed against the immunoregulatory plasma glycoprotein alpha 1-microglobulin were characterized. The MoAb were produced in mice immunized with a mixture of alpha 1-microglobulin homologues from man, guinea pig, rat and rabbit. Using radioimmunoassay, western blotting, affinity chromatography, and Scatchard analysis, the affinities and binding sites of the MoAb were analysed. All antibodies were more or less cross-reactive, but most showed a major specificity for one or two of the alpha 1-microglobulin homologues. None of the antibodies was directed against the carbohydrate moiety of alpha 1-microglobulin. Six of the MoAb had high affinity for the antigen and four of these were directed towards the same part of the molecule though differing in their species specificity. Five showed lower affinity for the antigen and were mainly directed towards epitopes on other parts of the molecule. Only some of the antibodies could block the proliferation of lymphocytes induced by human alpha 1-microglobulin. The blocking efficiency of the different antibodies was similar when tested on the stimulation of human or mouse lymphocytes, suggesting that the same part of the alpha 1-microglobulin molecule is responsible in both species. The magnitude of blocking by the different MoAb was not related to their affinities, emphasizing the importance of where on the alpha 1-microglobulin molecule, rather than how strongly, they bind. The binding of the strongest blocking antibody was shown to be directed to a C-terminal peptide of rat alpha 1-microglobulin, indicating that this part of alpha 1-microglobulin is important for the mitogenic effects. Thus the panel of anti-alpha 1-microglobulin MoAb should be a valuable tool for structural and functional studies of alpha 1-microglobulin.

Mitogenic effect of alpha 1-microglobulin on mouse lymphocytes. Evidence of T- and B-cell cooperation, B-cell proliferation, and a low-affinity receptor on mononuclear cells.

Human alpha 1-m microglobulin (alpha 1-m), a low molecular weight plasma protein, was found to exert mitogenic effects on mouse lymphocytes from lymph nodes and spleen. The stimulatory effects appeared to be strain-restricted: alpha 1-m induced a varying degree of proliferation of lymphocytes from three strains, whereas one strain responded poorly. Experiments with lymphocyte subpopulations showed only weak stimulatory effects of alpha 1-m on purified T and B lymphocytes cultivated alone. The addition of mitomycin-treated cells of the other subpopulation could not restore the proliferative responses in either T or B lymphocytes. Strong stimulations were recorded only when both T and B lymphocytes were present, indicating that the T and B lymphocytes cooperate to achieve the proliferation. However, FACS studies on cultured splenocytes indicated that the proliferating cells are predominantly B lymphocytes. These data extend our earlier findings of a mitogenic effect of alpha 1-m on guinea pig lymphocytes. Furthermore, results were obtained indicating the presence of a receptor on mononuclear cells. Iodine-labelled alpha 1-m was bound to mononuclear cells prepared from spleens, and the binding could be blocked by an excess of non-labelled alpha 1-m. Scatchard plotting of the data gave an equilibrium constant of 0.7 x 10(5)/M for the binding between alpha 1-m and the receptor. Together with the documented inhibitory activity of alpha 1-m on antigen-driven proliferation of lymphocytes, these results suggest an immunoregulatory role for alpha 1-m.

An intriguing member of the lipocalin protein family: alpha 1-microglobulin.

The plasma protein alpha 1-microglobulin is a member of the lipocalin protein superfamily. In the last few years, the work on alpha 1-microglobulin has given unexpected and promising new results. Of particular interest are its molecular association with immunoglobulin A and with proteinase inhibitors, and its interactions with the immune system.

Alpha 1-microglobulin is mitogenic to human peripheral blood lymphocytes. Regulation by both enhancing and suppressive serum factors.

Human alpha 1-microglobulin (alpha 1-m), a 26 kilodalton serum glycoprotein, was found to exert mitogenic effects on human peripheral blood lymphocytes (PBL) in serum-free medium. Purified T cells, but not B cells, responded with proliferation to alpha 1-m, but only in the presence of monocytes. The mitogenic activity could be partially neutralized by a mouse monoclonal antibody against alpha 1-m. The mitogenicity was species-specific, since alpha 1-m homologues from rats, guinea pigs and rabbits had no effect on human PBL. In a previous study, no effect of alpha 1-m was seen on PBL in the presence of 20% serum, and, therefore, we studied the influence of different concentrations of serum on the alpha 1-m-induced mitogenicity. Thus, human serum enhanced the mitogenic effects of alpha 1-m on human PBL at 1% concentration (v/v) and suppressed the effects at 10%. The suppressing effect of serum at 10%, but not the enhancing effect at 1%, seemed to be conserved among several species. To test the effect of serum proteins of different molecular sizes, human autologous serum was separated by gel chromatography on Sephadex G-200 into four fractions. Fractions 1 and 2 (roughly containing proteins larger than 100 kilodaltons) suppressed the mitogenic effects of alpha 1-m, while fractions 3 and 4 enhanced the stimulation by alpha 1-m, at 0.5% and concentrations above. It is concluded that the mitogenic effect of alpha 1-m on lymphocytes is regulated by several serum factors, both enhancing and suppressive, that does not have any proliferative effect of their own. It can be speculated that the balance between enhancing and suppressing co-factors in the blood determines the degree of the stimulation of lymphocytes by alpha 1-m. This is compatible with an immunomodulatory role for alpha 1-m, in spite of its relatively constant plasma levels in health and disease.

Islet transplantation to the renal subcapsular space improves late complications in streptozotocin-diabetic rats.

Transplantation of isolated islets is a promising approach in the treatment of diabetes. We have examined the long-term effects on the late complications of islet transplantation in an experimental diabetes model in the rat. Diabetes was induced by streptozotocin (70 mg/kg i.v.) and the rats were treated with either insulin (daily injection of 40 U) or transplantation of 1,000 freshly isolated, hand-picked, islets into the left renal subcapsular space. Both islet transplantation and insulin treatment completely normalized the increased levels of blood glucose, urine volume and water intake that were observed in the diabetic rats. The decreased growth rate of the diabetic rats was almost normalized by both treatment protocols. As for late complications, after 3 months, all untreated diabetic rats had cataract. They also had swelling and vacuolation of renal tubular cells, and, consistent with this, very high levels of urinary beta 2-microglobulin excretion. Both islet transplantation and insulin treatment completely prevented these late complications. Thus, islet transplantation to the renal subcapsular space is in this experimental model as good as insulin treatment in treating the clinical signs of diabetes and in preventing diabetic complications in the eye and kidney.

The complete amino acid sequence of rat beta 2-microglobulin.

The primary structure of rat beta 2-microglobulin (beta 2m) was determined. It is a polypeptide of 99 amino acids with the following sequence: IQKTPQIQVY SRHPPENGKP NFLNCYVSQF HPPQIEIELL KNGKKIPNIE MSDLSFSKDW SFYILAHTEF TPTETDVYAC RVKHVTLKEP KTVTWDRDM. The primary structure was determined by NH2-terminal sequence analysis together with sequence determination of one cyanogen bromide fragment and one tryptic peptide. Of other known beta 2m sequences, rat beta 2m is most homologous to mouse beta 2m (83% identity). The rabbit, human, and guinea pig sequences are more distant, with 24, 27, and 31% differences, respectively.

Structural relationship between alpha 1-microglobulin from man, guinea-pig, rat and rabbit.

Rabbit alpha 1-microglobulin was purified from the urine of sodium-chromate-treated animals by the use of gel chromatography on Sephadex G-100, affinity chromatography on concanavalin-A--Sepharose and ion-exchange chromatography on DEAE-Sephadex. Rabbit alpha 1-microglobulin had a molecular mass of 25.6 kDa on SDS/polyacrylamide gel electrophoresis. Alpha 1-microglobulin has previously been purified from the urine of humans, guinea-pigs and rats by similar methods, and the molecular masses of the four homologues were compared by SDS/polyacrylamide gel electrophoresis and gel chromatography in a denaturing medium. By these two methods the human homologue was 6 kDa and 3 kDa larger, respectively, than the other three proteins. Endoglycosidase F digestion of alpha 1-microglobulin, followed by SDS/polyacrylamide gel electrophoresis, revealed three protein bands in the human alpha 1-microglobulin sample, and only two bands in guinea-pig, rat and rabbit alpha 1-microglobulin, with a gap between each band of 2.6--2.9 kDa. The amino-terminal amino acid sequences of the four homologues were determined and between 72% and 81% homology was seen. The five amino-terminal amino acids present in the other species were missing in guinea-pig alpha 1-microglobulin. Our results indicate that human alpha 1-microglobulin is substituted with two N-linked oligosaccharides, while only one is attached to each of the other alpha 1-microglobulins, and that the extra glycosylamine-linked oligosaccharide in the human protein is attached to asparagine in position 17. Finally it is shown that all four homologues inhibit antigen stimulation of human lymphocytes, a finding which is consistent with our previous suggestion that the N-linked oligosaccharides carry the immunosuppressive activity of alpha 1-microglobulin.

Cross-reacting monoclonal anti-alpha 1-microglobulin antibodies produced by multi-species immunization and using protein G for the screening assay.

In order to generate monoclonal antibodies (MAb) directed against the low molecular weight glycoprotein alpha 1-microglobulin, a BALB/c mouse was immunized with a mixture of human, guinea pig, rat and rabbit alpha 1-microglobulin homologues (multi-species immunization) and boosted several times. On day 194, the mouse splenocytes were fused to SP2/0 myeloma cells. The resulting hybridomas were screened for anti-alpha 1-microglobulin activity against the alpha 1-microglobulin mixture or against the individual homologues. For this screening, protein G (the newly described IgG-binding streptococcal protein) was used in a solid-phase radioimmunoassay. The binding of protein G to immobilized antigen-antibody complexes was enhanced by pre-incubation with rabbit anti-mouse immunoglobulin G. The result was a panel of nine established hybridoma lines, all producing unique monoclonal antibodies, of IgG1 or IgG2a class, to alpha 1-microglobulin. The antibodies were not only reactive in solid-phase radioimmunoassay, but they could also immunoprecipitate 125I-labeled soluble alpha 1-microglobulin. Moreover, they reacted specifically with the alpha 1-microglobulin band in Western blots of urinary proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Such monoclonal antibodies are potentially valuable reagents for the further characterization of alpha 1-microglobulin.

Alpha 1-microglobulin is mitogenic for guinea pig lymphocytes.

Guinea pig alpha 1-microglobulin (alpha 1-m) was found to exert mitogenic effects on guinea pig lymphocytes, i.e. peritoneal exudate lymphocytes (PEL) or T lymphocytes from regional or mesenteric lymph nodes (LNL). On the other hand, spleen T lymphocytes did not respond to alpha 1-m. Stimulation of LNL required the presence of accessory cells. The stimulatory effect was strain-specific: alpha 1-m clearly induced proliferation of lymphocytes from Strain 2 guinea pigs, whereas Strain 13 lymphocytes responded poorly or not at all. Moreover, alpha 1-m inhibited antigen-driven proliferation of guinea pig lymphocytes. None of the effects described were species-specific, e.g. human alpha 1-m exerted similar effects on the guinea pig lymphocytes. These data indicate that alpha 1-m may be involved in the regulation of lymphocyte activation.