Action of allogeneic and syngeneic splenic extracts on primary cell cultures from inbred mice

The effect of allogeneic and syngeneic extracts from the spleens of male and female inbred mice on primary cultures of fibroblasts obtained from the subcutaneous connective tissues of fetuses of CBA and C57BL/6J mice was studied. The cytotoxic and growth-inhibiting action on the cultures was successively enhanced by the use of extracts from syngeneic male and allogeneic female and male tissues. Consequently, an increase in the degree of antigenic difference between the target cells and extracts led to enhancement of the phenomenon of allogeneic inhibition. It was shown for the first time that in a syngeneic system extracts from male tissues (containing the weak H-Y antigen) have a cytotoxic action on cells from female inbred mice, i.e., that they induce a reaction of the allogeneic inhibition type.Research Laboratory of Experimental Immunobiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR N. N. Zhukov-Verezhnikov.) Translated from Byulleten' Éksperimental'noi biologii i meditsiny, Vol. 86, No. 10, pp. 486–488, October, 1978.     

Dendritic cell number is related to IL-4 expression in the airways of atopic asthmatic subjects

BACKGROUND: Airway dendritic cells are essential for stimulating naive T cells in response to inhaled antigen and for the development of allergic sensitization. IL-4 in vitro can distinguish dendritic cell lines from peripheral blood mononuclear cells. Our study had the following aims: 1) to compare the distribution of CD1a+ dendritic cells and IL-4+ cells, in the bronchial mucosa of asthmatics and controls 2) to determine the relationship between the numbers of CD1a+ dendritic cells and IL-4+ cells in the bronchial mucosa of asthmatics 3) to determine whether CD1a+ cells express the IL-4 receptor. METHODS: Twenty atopic asthmatic and eight normal subjects were studied. In each subject, bronchoscopy with bronchial biopsies was performed. CD1a, IL-4, and IL-4 receptor expressions were evaluated by immunohistochemistry. RESULTS: The number of CD1a+ and IL-4+ cells was significantly higher in asthmatics than controls. The number of CD1a+ cells was positively correlated to the number of IL-4 + cells. Bronchial biopsy serial section studies showed that CD1a+ cells express the receptor for IL-4. CONCLUSIONS: These results suggest that an increased amount of IL-4 may play a physiopathologic role in maintaining the dendritic cell pool in vivo. Therefore, because of possible IL-4 activity on antigen-presenting cells in T-cell immune responses to allergens, an important new role of IL-4 in asthma inflammation can be envisaged.     

Anti-neutrophil cytoplasmic antibodies (ANCA) in ulcerative colitis: anti-cathepsin G and a novel antibody correlate with a refractory type

We analysed the clinical significance of ANCA in patients with ulcerative colitis. On either an indirect immunofluorescence assay or an ELISA with fixed neutrophils, 71% (25/35) of the patients were positive for ANCA. However, only half of them reacted with either cathepsin G or lactoferrin. Western blot assays revealed positive bands in 40% (10/25) of the antibody-positive patients. The sizes of the bands detected were ≈58, 47, 44, 40, and 28 kD. No significant correlation was found between the ANCA positivity and various variables, i.e. disease activity, extent of lesion, or treatment of the disease. The anti-cathepsin G and 28-kD positivity, however, significantly correlated with a refractory type of ulcerative colitis.     

Immunoelectron microscopic localization of carcinoembryonic antigen in gastric adenocarcinoma cell lines

The distribution of carcinoembryonic antigen (CEA) in human gastric adenocarcinoma cell lines (HPE-GAC-3 cells and HPE-GAC-2 cells) was determined immunohistochemically by indirect peroxidase-labeled antibody method at the light and electron microscopic levels. In GAC-3 cells that proliferated as non-adherent single cells, CEA was located in the perinuclear spaces, the endoplasmic reticulum, Golgi apparatus, vesicles, multivesicular body (MVB) and entire plasma membrane. Membrane CEA was shown to be internalized into MVB in GAC-3 cells. In GAC-2 cells that form an acinus, CEA was predominantly present along the microvilli of the lumina) surface and in glycocalyceal bodies, the vesicles which bud from the microvilli into the lumen. These results suggest that in poorly differentiated cancer cells CEA is transported over the entire cell surface, retained on the membrane and accumulated Into the cell by way of the MVB, but in well differentiated cancer cells the newly synthesized CEA is rapidly and predominantly transported to the luminal surface and rapidly released from the membrane into the lumen by way of the glycocalyceal body.     

Conjugation of DNA fragments to protein carriers by glutaraldehyde: immunogenicity of oligonucleotide-hemocyanin conjugates

The practical realization of the concept of specific immunotherapy for systemic lupus erythematosus (SLE) has been hampered, thus far, by an inability to link DNA fragments to carrier protein. In this paper, a novel technique is described, in which glutaraldehyde is the linking agent. A 2-stage method was used to link oligonucleotides to a soluble protein carrier, such as keyhole limpet hemocyanin (KLH) or human gamma globulin (HGG), whereas a 1-stage technique was sufficient to link oligonucleotides to sheep red cells. Both the ultraviolet absorbance spectrum and diphenylamine assay demonstrated that oligonucleotides were coupled to soluble protein. The conjugate of oligonucleotide to protein carrier appears to be recognized by anti-DNA antibody since oligonucleotide linked to either KLH or HGG inhibited the binding of anti-DNA antibody in vitro, and oligonucleotide-coupled sheep cells are agglutinating by seropositve sera from lupus patients. In addition, oligonucleotide-KLH raised hemagglutinating antibody to denatured DNA in C57BL/6, DBA/2 or NZB mice, as well as IgG antibody as detected by SPRIA in C57BL/6 and DBA/2 mice. The significance of this new method for the development of an antigen specific therapy of SLE is discussed.     

Preferential presentation of herpes simplex virus T-cell antigen by HLA DQA1*0501/DQB1*0201 in comparison to HLA DQA1*0201/DQB1*0201

The HLA DQA1 locus is polymorphic. Haplotypes containing HLA DQA1*0501, but not HLA DQA1*0201, together with HLA DQB1*0201 are associated with Grave's disease and celiac sprue. In this report, we demonstrate a functional correlate of DQA1 polymorphism. T cells infiltrating a herpes simplex virus (HSV) lesion from a HLA DQ 2,7 individual yielded a virusspecific CD4+ clone restricted by DQ2. Presentation of viral peptide and protein segregated with DQA1 allele, because cell lines bearing DQA1*0501/DQB1*0201 heterodimers presented antigen in proliferation and cytotoxicity assays much more efficiently than cell lines bearing DQA1*0201/DQB1*0201. Binding of viral peptide to cell lines bearing DQA1*0201, in comparison to DQA1*0501, was only moderately reduced and may not explain this effect. Truncation and substitution analyses of peptide binding and T-cell activation were performed to determine which viral peptide residues contacting TCR might therefore be presented in an altered conformation by DQA1*0201/DQB1*0201. Residues 432, 435, 437, 438, and 440 (position P1, P4, P6, P7, and P9) contributed to DQ2 binding, whereas residues 431, 433, 434, and 436 (positions P-1, P2, P3, and P5) contributed to TCR contact. Differential presentation of peptide by HLA DQ2 heterodimers varying at the DQA1 locus may have relevance to host defense and the pathogenesis of HLA DQ2-associated autoimmune diseases. Human Immunology 53, 195-205 (1997).     

Inhibition of endogenous carcinoembryonic antigen (CEA) increases the apoptotic rate of colon cancer cells and inhibits metastatic tumor growth

It has been suggested that carcinoembryonic antigen (CEA) enhances metastatic seeding of colon cancer cells due to its homo- and heterophilic binding properties. Our recent finding that endogenous CEA protects colon cancer cells against apoptosis suggests a more complex role of CEA in cancer progression. In this study we compared the in vitro effects of endogenous CEA on tumor cell aggregation and cell cycle regulation of human HT29 colon cancer cells with the corresponding in vivo effects, i.e. tumor cell seeding and formation of metastatic lesions. Stable expression of CEA targeted ribozymes (Rz) under control of a tet-off promoter system allowed regulation of CEA levels on the mRNA and protein level by 50%. Downregulation of CEA levels inhibited tumor cell aggregation by 70%. In accordance with previous studies [1], reduction of CEA levels increased in vitro the apoptotic rate and reduced colony formation by 30% to 50%. To determine the in vivo effect of CEA-dependent aggregate formation and its growth regulating role under apoptotic stress, HT29 cells with high and low CEA levels, respectively, were injected into nude mice. Immunostaining of lung microsections revealed similar numbers of tumor cells one hour after injection. 24 h later virtually all cells were removed from the lung in both groups. However, after 6 weeks all doxycycline treated mice (Rz off = CEA high) showed 14.5 ± 4.6 metastatic lung lesions/mouse while 0.2 ± 0.2 lesions/mouse appeared in the untreated group (Rz on = CEA low) ( P<0.001). Our study demonstrates a multifunctional role of CEA and indicates a prometastatic role of CEA independent of its adhesive function possibly due to its anti-apoptotic function. This revised version was published online in July 2006 with corrections to the Cover Date.     

H-Y Antigen Expression Patterns in Human X- and Y-Chromosome-Bearing Spermatozoa

PROBLEM: Restricted expression of H-Y antigen on Y-chromosome-bearing sperm has been reported in some species, although such preferential expression for H-Y antigen in human sperm has yet to be described. In this study, an immunomagnetic approach was used to characterize antigen expression patterns as a function of sex-chromosome content. METHOD OF STUDY: Human sperm was treated with monoclonal immunoglobulin (Ig) M antibodies directed against H-Y antigen. This preparation then was incubated with sheep antimouse IgM antibody affixed to paramagnetic beads, which then were exposed to a magnetic field and sorted. X- and Y-chromosome frequencies in the two subgroups of sperm were assayed by multiprobe fluorescent in situ hybridization (FISH). RESULTS: Sperm were immunomagnetically separated into two populations: a reactive group (presumably, H-Y Ag+); and a nonreactive group (presumably, H-Y Ag-). Triple-color FISH analysis of 1,600 spermatozoa (800 in each group) showed the antigen's expression to be somewhat more prevalent among Y-chromosome-bearing sperm (54.1%), but a large proportion of Y-chromosome-bearing sperm (49.0%) did not express this antigen. The difference was not significant (P = 0.43). CONCLUSIONS: The expression of H-Y antigen has a slightly higher frequency in human sperm containing the Y-chromosome, but its expression among X-chromosome-bearing sperm also is considerable. Current immunologic techniques relying on this antigen are unlikely to effect the sex selection of human sperm.     

A proportion of proteinase 3 (PR3)-specific anti-neutrophil cytoplasmic antibodies (ANCA) only react with PR3 after cleavage of its N-terminal activation dipeptide

ANCA directed against PR3 are highly specific for Wegener's granulomatosis and microscopic polyangiitis, and have been implicated in the pathogenesis of small vessel vasculitis. Most PR3-ANCA are directed against conformational epitopes on PR3. This study was designed to determine whether the cleavage of the N-terminal activation dipeptide of PR3 is required for the binding of PR3-ANCA. Recombinant PR3 (rPR3) variants were expressed in the epithelial cell line, 293. As confirmed by radiosequencing, the rPR3 secreted into the 293 cell culture supernatant is N-terminally unprocessed. Two enzymatically inactive rPR3 mutants were expressed in 293 cells: rPR3-S176A and δ -rPR3-S176A. rPR3-S176A contains the N-propetide Ala-2-Glu-1, δ -rPR3-S176A does not. Culture supernatants of rPR3-S176A and δ -rPR3-S176A expressing 293 cells were used as sources of target antigen for PR3-ANCA testing by capture ELISA. Forty unselected consecutive PR3-ANCA⁺ sera were tested. With δ -rPR3-S176A as antigen all 40 were recognized, compared with only 34 of 40 when rPR3-S176A served as target antigen. The majority of the serum samples contained a mixture of antibodies reacting with epitopes accessible on the mature and on the proform of PR3. In conclusion, the cleavage of the N-terminal activation dipeptide of PR3 is not an absolute requirement for recognition by all PR3-ANCA. However, a substantial proportion of PR3-ANCA recognize (a) target antigen(s) exposed only after the conformational change of PR3 associated with the N-terminal processing. In 15% of sera this PR3-ANCA subset occurred exclusively. PR3-ANCA subtypes can be differentiated using specifically designed rPR3 variants as target antigens, and non-haematopoietic mammalian cells without regulated secretory pathway can be used for their expression.     

Genetic markers in the Tuvan population of Todja, Siberia

Todja is a secluded region of northern Tuva situated in the Sayany Mountains, Siberia. The aboriginal population of Todja is Tuvan. A total of 128 healthy Tuvans living in Todja were typed for HLA-A, -B and -C antigens and several plasma and erythrocyte protein polymorphisms (Hp, Tf, Gc, ESD, ACP, PGM1, PGD and ADA). The observed frequencies of all 8 blood protein and HLA genotypes were in agreement with Hardy-Weinberg expectations. The most frequent HLA antigens in Todjans are A2 (0.36). A3 (0,24), A9 (0.50), B15 (0.34) and B40 (0.50). HLA haplotypes A2B5, A2B40, A9B15 and A9B40 are most common in this population. The observed frequencies of protein polymorphisms and HLA antigens and haplotypes in Todjans are similar to those of other Mongoloid populations. A comparison of HLA frequencies currently observed in Todjans with those obtained 20 years ago at the same locality showed minor changes attributable to the effect of migration.