A method for blocking antigen-independent binding of human IgM to frozen tissue sections when screening human hybridoma antibodies

Using an indirect immunoperoxidase technique antigen independent binding of both human monoclonal and polyclonal IgM was found to a wide range of frozen sections of normal and malignant human glandular epithelia. Identical binding was found using dimeric human IgA (dIgA), whereas no binding was found with monomeric human IgA or human IgG. Secretory component (SC) was found to be the component in these tissues mediating antigen-independent binding of human IgM and dIgA antibodies. A method for the blocking of this antigen-independent binding of human IgM and dIgA was evaluated. Frozen sections of tissues containing SC were blocked with antibody to endogenous immunoglobulin and preincubated with rabbit anti-secretory component antibody (anti-SC) before applying the human monoclonal antibody. This treatment blocked the binding of control polyclonal and monoclonal human IgM to sections of SC-containing tissues such as respiratory and colonic epithelia. The influence of anti-SC on the binding of dIgA could not be established due to interactions between the anti-SC antibody and the IgA preparation. Using this method human hybridoma supernatants containing IgM could be readily screened for reactivity with frozen tissue sections from patients with colo-rectal cancer. This approach is recommended for the screening of human IgM monoclonal antibodies on frozen human tissue sections.     

Normal newborn and adult human lung collagen--analysis of types.

Previous work has shown in experimental animals that lung contains extractable collagen of types I and II and possibly III and IV. The current studies describe the types of collagen which are found in normal newborn and adult human lung. Autopsy lung specimens were dissected into tissue which was cartilage free and tissue which contained cartilage. Pleura was excluded. Extraction of distal lung which did not contain cartilage by using 1.0 M NaCl and 0.5 N acetic yielded collagen having the characteristics on acrylamide gel electrophoresis and CM-cellulose chromatography of type I collagen [alpha 1(I)] alpha2. Newborn and adult type I lung collagen are similar as compared by amino acid analysis and cyanogen bromide peptide fragment analysis. Furthermore, distal lung type I collagen is very similar, if not identical, to that found in human skin. Human bronchial cartilage collagen is similar to that found in other human cartilages. Type II collagen, [alpha 1(III)], can be extracted from human distal lung using pepsin digestion and differential salt precipitation at 1.85 M sodium chloride. This distal lung type III collagen appears identical with that found in human skin. These data support the concept that normal human lung contains collagen of types I, II, and III, identical to that found elsewhere in the body. Current techniques have not revealed a unique lung collagen. This should lead to the studies which will investigate the types and ratios of the collagens in diseased human lung.     

Monocyte activating factor in sarcoidosis. II. Secretion of the factor from monocytes

The normal human monocytes pretreated with the monocyte activating factor found in sarcoidosis sera were shown to secrete a factor which induced normal human monocytes to spread as well as to increase their cell size. Phagocytosis and glucose consumption of normal human monocytes were also increased by this secondarily obtained factor. Its molecular weight was about 70,000. These results indicate that this secondary factor may be the same substance as the monocyte activating factor found in sarcoidosis sera. The normal human monocytes pretreated with the monocyte activating factor were also shown to liberate a factor which generated macrophage migration inhibitory factor in cooperation with normal human sera.     

Fungus-like hyphochytrids associated with human disease.

We report two cases, with liver and brain abscess, respectively, where fungus-like organisms belonging to the Hyphochytriomycota were found at the site of inflammation together with Peptococcus in the first and Cysticercus cellulosae in the second case. This is the first time these groups of organisms have been reported in human material. The role of hyphochytrids in human pathology remained uncertain as they were found together with already known human pathogens.     

The Cardiac Autoimmune System: VII. Heart Specific Auto-Antibodies Produced in Rabbits Immunized with Human Heart

Rabbit antiserum to human heart, which revealed at least twelve cardiac “tissue” antigens after absorption with human plasma, was further absorbed with human kidney and skeletal muscle. The resulting antiserum reacted only with human heart extracts in immunodiffusion tests. One antigen restricted to heart tissue was found regularly, while a second one was visualized occasionally. The former was also detected in extracts of rabbit and rat hearts. The protein involved was identified as auto-antigen B, as seen earlier, in the complex patterns found with rabbit anti-rabbit heart auto-antibodies. “Tteactions of identity” were also found with rat anti-rat heart auto-antibodies. The same immune system was seen with several other heterologous anti-heart sera, including duck anti-rabbit heart. The auto-antigen B protein was found to have a molecular weight of about 160,000 and an isoelectric point of pH 5.2.     

Legionella pneumophila utilizes the same genes to multiply within Acanthamoeba castellanii and human macrophages

In previous reports we described a 22-kb Legionella pneumophila chromosomal locus containing 18 genes. Thirteen of these genes (icmT, -R, -Q, -P, -O, -M, -L, -K, -E, -C, -D, -J, and -B) were found to be completely required for intracellular growth and killing of human macrophages. Three genes (icmS, -G, and -F) were found to be partially required, and two genes (lphA and tphA) were found to be dispensable for intracellular growth and killing of human macrophages. Here, we analyzed the requirement of these genes for intracellular growth in the protozoan host Acanthamoeba castellanii, a well-established important environmental host of L. pneumophila. We found that all the genes that are completely required for intracellular growth in human macrophages are also completely required for intracellular growth in A. castellanii. However, the genes that are partially required for intracellular growth in human macrophages are completely required for intracellular growth in A. castellanii. In addition, the lphA gene, which was shown to be dispensable for intracellular growth in human macrophages, is partially required for intracellular growth in A. castellanii. Our results indicate that L. pneumophila utilizes the same genes to grow intracellularly in both human macrophages and amoebae.     

Spontaneous production of human gamma interferon in vitro by splenic lymphocytes of patients with idiopathic thrombocytopenic purpura.

Lymphocytes derived from spleens of traffic trauma victims do not appear to produce human interferon (IFN) activity, spontaneously, in vitro. However, lymphocytes derived from spleens of four ITP patients were found to produce significant amounts of human IFN activity. The IFN activity produced by the splenic lymphocytes of ITP patients was neutralized by anti-gamma-IFN antisera but not anti-alpha or anti-beta antisera. The IFN activity was found to be unstable at pH 2.0 and at 56 degrees C. Thus the human IFN activity of splenic lymphocytes is characterized as human gamma-IFN. No human IFN activity was detectable in the serum of the ITP patients and it is not known whether the splenic lymphocytes of ITP patients also produce human gamma-IFN in vivo. The observations suggest that conditions prevail in the ITP state that predispose the splenic lymphocytes to produce human gamma-IFN without stimulation by exogenously added inducer.     

Immunoglobulin production in severe combined immunodeficient (SCID) mice reconstituted with human peripheral blood mononuclear cells.

Immunodeficient C.B-17 scid/scid (SCID) mice were reconstituted with human peripheral blood mononuclear cells and analyzed for humoral immunity. The majority of adoptively transferred animals had serum levels of 1-4 mg/ml of human IgG 8-12 weeks after i.p. reconstitution with 20 x 10(6) PBMC, whereas the IgM and especially IgA concentrations were considerably lower. The half-lives of human IgG, IgM, and IgA in SCID mice were 12 days, 36, and 23 hours, respectively. Furthermore, IgA was rapidly secreted into bile indicating that the low IgA concentration was mainly caused by a high turnover rate. IgG subclass distribution in mouse serum was similar to that found in the donor serum. Irradiation with 3 Gy prior to adoptive transfer resulted in increased levels of human IgG early after reconstitution, whereas both IgM and IgA concentrations were impaired. A polyclonal serum Ig pattern was found 4 weeks after transfer of human cells later frequently followed by a predominance of oligoclonal bands. Unexpectedly, oligoclonal bands were also found using donors with a negative Epstein-Barr virus serology. Human cells were found to reside in the peritoneal cavity for several months. Within 2 weeks of reconstitution, human cells were also identified in lymphoid structures in the vicinity of the liver hilus with a later spread to other lymphoid organs. Homing of human cells to skin and gut was not seen.     

Ion chromatographic method for simultaneous determination of nitrate and nitrite in human saliva

A simple, rapid, accurate and sensitive method is proposed for the simultaneous determination of nitrite and nitrate in human saliva. Nitrite and nitrate present in the human saliva were determined after 10- to 100-fold dilution with ion chromatography (IC) using suppressed conductivity detection. Recoveries of nitrite and nitrate were found to be ranged between 95% and 101%. The method was linear (r2=0.9991) over the concentration working range. The detection limits were found to be 15.0 microg/l and 33.5 microg/l, for nitrite and nitrate, respectively. Ions that are present in human saliva and several other ions that are suspected to affect nitrite and nitrate determination were checked. It was found that most of the ions did not cause any interference in the determination. The method allows simultaneous determination of nitrite and nitrate in human saliva.     

Morphological differences in Neisseria meningitidis pili.

Disease and carrier isolates of Neisseria meningitidis were examined for their ability to adhere to human buccal epithelial cells and human cell lines and to hemagglutinate human erythrocytes, properties thought to be associated with the presence of pili. Seventy percent (7 of 10) of carrier isolates were found to be highly adherent to human buccal epithelial cells and to agglutinate human A, B, O, Rh-, and Rh+ erythrocytes. In contrast, 60% of the disease isolates adhered poorly to human buccal epithelial cells and 80% failed to agglutinate human erythrocytes. No adherence of either disease or carrier isolates was observed when several human cell lines were tested. When the meningococcal strains were examined by electron microscopy, 7 of 10 disease isolates were found to possess large bundles of aggregated pili (alpha-type pili), while 7 of 10 carrier isolates were found to have numerous unaggregated pili (beta-type pili). A monoclonal antibody against meningococcal pili and one against gonococcal pili reacted with 6 of 10 piliated carrier isolates and 4 of 10 piliated disease isolates. These results suggest that meningococci, like gonococci, possess different types of pili which differ in morphological, antigenic, and binding properties. In addition, antigenic and morphological differences between pili from carrier and disease isolates were observed as well as differences in adherence and hemagglutinating properties.     

Sequences and repertoire of the human T cell receptor alpha and beta chain variable region genes in thymocytes

To compare and contrast the human T cell antigen receptor (TcR) alpha and beta chain messages found in human thymocytes to those previously isolated from human peripheral blood T lymphocytes and other nonthymic sources, 13 TcR alpha and 13 TcR beta cDNA were isolated from a human thymocyte library and the nucleotide sequences were determined. The data indicate that, as was found in the peripheral T lymphocytes, the majority of the TcR alpha and TcR beta chain thymocyte cDNA were derived from potentially functional messages. Although the thymocyte-derived TcR cDNA do not contain any unique structural features when compared to TcR cDNA from mature T lymphocytes, 4 new J alpha segments, 17 new V-gene segments (9 V alpha; 8 V beta) and 7 additional V-gene families (4 V alpha and 3 V beta) and sequences had been identified. The exon C beta O, found in many murine thymocyte TcR beta messages, was not found in over 75 human beta chain messages. Based on these new data, a revised estimate of human TcR V alpha, J alpha and V beta repertoires is calculated. The most significant change has been the increase in the estimated number of human TcR V beta-gene segments to a total of about 100 distributed among about 18 families. The V alpha families are now revised upward to 16, with a total number of V alpha segments of 50. The estimate of the J alpha segments in humans remains between 50-100.     

A radioimmunoassay of human leukocyte interferon using protein A-containing Staphylococcus aureus

We report the establishment of a radioimmune assay for human alpha-interferon using highly purified alpha-interferon labeled with iodine-125, a purified rabbit immunoglobulin directed against human interferon and the Cowan strain of Staphylococcus aureus. Radiolabeling conditions used do not change the antigenicity of interferon molecules. The regression of counts bound upon log. dose was found to be linear down to 10 units/ml of alpha-interferon (6-7 X10-12 M). This assay was specific for alpha-interferons derived from human peripheral blood leukocytes and from a continuous line of lymphoblastoid cells. No cross-reaction was found with either human beta-interferon or murine interferon. Neither human serum nor plasma interfered with the assay. Correlation between biological assay and radioimmune assay was found to be significant.     

Parainfluenza 1 virus and multiple sclerosis: The conversion of 6/94 virus released from human brain cells and other mammalian cells into and infectious form by passage in macrophages

The parainfluenza type-1 6/94 virus isolated from lysolecithin-fused CV-1 and multiple sclerosis brain cells and propagated in eggs was found to infect human brain, and other mammalian cells. The virus obtained from the extracellular fluids of 6/94-infected human brain and MDBK cells was not infectious for a second passage in these cells but did replicate in cultures of mouse macrophages (IC-21). 6/94 virus obtained from infected macrophage culture fluid was in turn infectious for human brain and MDBK cells but the virus produced by the latter cultures was again found to infect only macrophages. Following treatment with trypsin, the noninfectious virus obtained from either human brain of MDBK cell culture fluids was found to possess restored infectivity. Ammo acid and carbohydrate radiolabeled 6/94 virus purified from MDBK and human brain cell culture fluid was examined by SDS-polyacrylamide gel electrophoresis before and after treatment with trypsin. The pattern, number and molecular weights of the viral polypeptides were compared to egg grown 6/94 virus and correlated with the restoration of infectivity for MDBK and human brain cells.     

Monoclonal anti-la antibody derived from a mouse with experimental SLE is similar to human anti-la antibodies

The La antigen is a highly conserved protein, originally defined by sera of patients with Sj?gren's syndrome or systemic lupus erythematosus (SLE). In the present study, we have produced and characterized a monoclonal anti-la antibody derived from mice with experimental SLE. The induction of SLE in these mice was achieved by their immunization with a murine monoclonal anti-idiotypic antibody against a common idiotype (16/6 Id) found in SLE patients. The monoclonal anti-La antibody derived from these mice was found to be virtually identical to the anti-La antibodies found in human autoimmune sera. First, its binding to different nuclear extracts, as well as to protease-digested HeLa nuclear protein extracts, was found to be identical to that of human anti-La antibodies. Second, an inhibition study on blotted proteins demonstrated a very close relationship between the epitopes recognized by the murine monoclonal anti-La antibody and the human anti-La serum. Third, the monoclonal anti-La antibody was found by immunofluorescence to be directed against a nuclear antigen that gave a speckled pattern. Finally, the monoclonal anti-La antibody immunoprecipitated the La-associated small RNAs. This report provides evidence for the similarity of murine anti-La antibody produced in experimental SLE and human anti-La antibodies formed in autoimmunity.     

Construction and expression of recombinant plasmids encoding type 1 or D-mannose-resistant pili from a urinary tract infection Escherichia coli isolate.

Isolates of Escherichia coli from human urinary tract infections frequently express adherence properties found less often among normal intestinal isolates. These properties include adherence to human uroepithelial cells and primary monkey kidney cells, as well as D-mannose-resistant hemagglutination of human erythrocytes, and they are mediated by a pilus type different from type 1. The genes encoding this pilus type (pyelonephritis-associated pili, pap) and those encoding type 1 pili have been cloned from a urinary tract infection isolate of E. coli and transferred to an E. coli K-12 derivative. The recombinant plasmids were found to express functional pili and to endow the new host with all of the adherence properties of the urinary tract infection isolate. Both pilus types were found to be genetically distinct, and unlike the adherence genes from bovine, porcine, and human diarrheal isolates, both were found to be chromosomally encoded.     

Immunological similarities between an experimental autoimmune myasthenia gravis model and human myasthenia gravis

The induction of experimental autoimmune myasthenia gravis (EAMG) in rabbits after immunization with an acetylcholine (ACh) conjugate was found to possess immunological similarities with human myasthenia gravis. Anti-ACh antibodies, present in human sera, recognized the antigenic determinant, glutarylcholine, used to raise anti-ACh antibodies in rabbits. Identification of anti-anti-ACh antibodies in MG patients enabled us to test for recognition of the anti-ACh antibodies present in rabbit sera. The reverse, the recognition of rabbit auto-anti-anti-ACh antibodies by human anti-ACh antibodies was also tested and found to be specific.     

A protein reminiscent of the epidermal SH-protease inhibitor occurs in squamous epithelia of man and rat.

The occurence of the human and rat epidermal SH-protease inhibitors in various human and rat tissues was studied by double radial immunodiffusion against specific antisera to the inhibitors. An immunoreactive protein was found in the extracts prepared from human and rat epidermis and from eosophageal and vaginal squamous epithelia, and from rat pro-ventricular squamous epithelium. No immunoreactive protein was found in man or rat in any other of their tissues, studied by us. The results strongly suggest that a protein reminiscent of the human or rat epidermal SH-protease inhibitor is present in squamous epithelia but not in other tissues. The identity of the epidermal inhibitor and the immunoreactive protein in the other squamous epithelia was confirmed by immunodiffusion, immunoelectrophoresis and gel chromatography, and by immunoinhibition of the papain inhibiting activity of the human epidermal and oesophageal inhibitors by gammaglobulins separated from antiserum to the human epidermal inhibitor.     

Distribution of serotypes and antimicrobial resistance genes among Streptococcus agalactiae isolates from bovine and human hosts

To better understand the emergence and transmission of antibiotic-resistant Streptococcus agalactiae, we compared phenotypic and genotypic characteristics of 52 human and 83 bovine S. agalactiae isolates. Serotypes found among isolates from human hosts included V (48.1%), III (19.2%), Ia and Ib (13.5% each), and II (5.8%). Among isolates from bovine hosts, molecular serotypes III and II were predominant (53 and 14.5%, respectively). Four and 21 different ribotypes were found among human and bovine isolates, respectively. A combination of ribotyping and serotyping showed that two bovine isolates were indistinguishable from human isolates. Resistance to tetracycline and erythromycin was more common among human (84.6% and 26.9%, respectively) than bovine (14.5% and 3.6%, respectively) isolates. tetM was found in all tetracycline-resistant human isolates, while tetO was the predominant resistance gene among bovine isolates. tet genes were found among various ribotypes. ermB, ermTR, and mefA were detected among erythromycin-resistant human isolates, while ermB was the only erythromycin resistance determinant among isolates from bovine hosts. For isolates from human hosts, erythromycin resistance genes appeared to be associated with specific ribotypes. We conclude that (i) human and bovine S. agalactiae isolates represent distinct populations; (ii) human host-associated S. agalactiae subtypes may occasionally be transmitted to bovines; (iii) while emergence of erythromycin and tetracycline resistance appears to largely occur independently among human and bovine isolates, occasional cross-species transfer of resistant strains or transmission of resistance genes between human- and bovine-associated subtypes may occur; and (iv) dissemination of antibiotic-resistant S. agalactiae appears to include both clonal spread of resistant strains as well as horizontal gene transfer.     

Rh antibodies produced by an isoimmunized chimpanzee; reciprocal relationship between chimpanzee simian-type isoimmune sera and human anti-Rho reagents.

An isoimmune chimpanzee serum was found to contain, in addition to expected antibodies of the simian-type specificity anti-Lc, a fraction of anti-Rho specificity, as proved by parallel tests with standard human anti-Rho sera as well as by absorption experiments using human and chimpanzee red cells. On the other hand, the two human anti-Rho sera tested in parallel were found to contain not only antibodies of specificity anti-Rho but also antibodies capable of detecting Lc specificity on the chimpanzee red cells. The reciprocal relationship between human and chimpanzee isoimmune sera constitutes the first example of symmetrical cross-reactivity between closely related species. The new source of anti-Rho reagents is expected to contribute additional information on the human Rh-Hr blood group system.