A base substitution in the interleukin-10 (IL-10) promoter between Sp1 and ets-1 binding sites is not associated with variation of IL-10 levels.

Interleukin 10 (IL-10) may play an important anti-inflammatory and immunoregulatory role in asthma. In this study, we investigated the role of a C to A substitution at position -627 of the IL-10 promoter, located in a necessary transcriptional region, which contains a number of putative transcriptional binding sites. The -627 nucleotide position is itself flanked by Sp-1 and ets-1 binding sites. We studied the allele frequency in 53 unrelated subjects from an admixed Caucasian, Asian and Pacific Islander group with personal or family histories of asthma. The frequency of homozygous C/C, heterozygous C/A, and homozygous A/A alleles at position -627 was 0.28, 0.44 and 0.28, respectively. In vitro assays indicated no differences between the C/C and A/A forms in binding transcriptional factors, especially Sp-1 factor, or in promoter activity. Moreover, in this selected population, there was no association between the C to A substitution and serum IL-10 levels. The mean level of IL-10 serum was determined to be 3.87 +/- 1.23 pg/ml in subjects carrying the A/A genotype, 3.47 +/- 0.57 for C/C genotype and 3.13 +/- 0.41 for the heterozygous (C/A genotype). This requires confirmation by comparing to non-asthmatic subjects. We conclude that although the -627 A allele occurs frequently (50% of alleles) in this selected group, in vitro assays and serum IL-10 levels suggest that the -627C-->A substitution represents a silent or neutral variant in the IL-10 promoter.

Do urinary mononuclear cells reflect disease activity in lupus nephritis?

Glomerular inflammation is associated with urinary mononuclear cells (UMC) in a number of diseases including IgA nephropathy and glomerulonephritis. We examined UMC from children with lupus nephritis for a number of years to characterize the types of mononuclear cells found in urine and to determine if they were associated with active lupus nephritis. Detailed analysis of UMC by cell counts and by flow cytometry showed that monocytes were the clearly dominant cell type. Evaluation of the smaller number of lymphocytes found in the urine of patients with active lupus nephritis demonstrated a strong predominance of CD8+ lymphocytes, in contrast to the normal CD4+/CD8+ ratio that is found in peripheral blood. The degree of proteinuria strongly correlated with the presence of UMC. The UMC counts decreased as their clinical condition improved as indicated by lower indices of flare. These observations suggest that UMC may be a valuable tool in detecting and monitoring disease activity in patients with severe lupus nephritis. More importantly, this study indicated that both monocytes and cytotoxic CD8+ T cells may play a role in pathogenesis of lupus nephritis.

Cytokine mRNA repertoire of articular chondrocytes from arthritic patients, infants, and neonatal mice.

Articular chondrocytes from nine arthritic patients, five infants, and Balb/c neonatal mice were analyzed for the presence of various cytokine mRNAs by a reverse transcriptase polymerase chain reaction (RT-PCR). Four cytokine mRNAs, interleukin (IL)-6, IL-8, IL-11, and macrophage colony stimulating factor (M-CSF), were detected in all human chondrocytes, regardless of source. IL-10, IL-12p35, and tumor necrosis factor alpha (TNF-alpha) transcripts were found in at least 12 of the 14 human samples. IL-13, granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), and TNF-beta mRNAs were found more predominantly in infant samples and in samples from patients with rheumatoid arthritis (RA) compared with samples from patients with osteoarthritis (OA). Another group of cytokine mRNAs, IL-1 (alpha, beta), IL-4, IL-5, and IL-7, were only weakly expressed in some human samples. The cytokine transcripts that were not found were IL-2, IL3, and interferon gamma (IFN-gamma). Because of the large array of cytokine transcripts detected, human chondrocyte preparations were further purified by reacting them with a monoclonal antibody specific to chondrocyte differentiation antigen and subjecting them to fluorescent-activated cell sorting. A similar array of cytokines was found between the sorted and unsorted chondrocytes, although TNF-alpha, G-CSF and GM-CSF transcripts appeared to be upregulated during the sorting process. Human chondrocytes that dedifferentiated into fibroblasts (a 40-day and a 77-day culture) no longer expressed mRNAs for IL-1, G-CSF, GM-CSF, and TNF-alpha, but all other cytokine mRNAs remained detectable. Although certain phenotypic characteristics were lost, including reactivity to chondrocyte-specific monoclonal antibodies and morphological features, chondrocytes in long-term culture still expressed cytokine mRNAs. As expected, more consistent results were obtained when seven preparations of chondrocytes from neonatal Balb/c mice were examined using available cytokine primers. They contained IL-1, IL-5, IL-6, IL-7, IL-12, GM-CSF, M-CSF, transforming growth factor beta (TGF-beta), TNF-alpha, and TNF-beta mRNAs but lacked IL-2, IL-3, IL-4, IL-10, and IFN-gamma mRNAs. Future experiments to define conditions by which these cytokine protein products are expressed are needed to help assess their roles in chondrocyte biology and in disease states.

Differentiation antigens of human articular chondrocytes and their tissue distribution as assessed by monoclonal antibodies.

Five monoclonal antibodies (HuMC1, HuMC2, HuMC3, HuMC4 and HuMC5) raised against intact human articular chondrocytes were tested with chondrocyte samples from arthritic and non-arthritic patients by surface immunofluorescence and by immunoperoxidase labeling of fixed cells. All acetone-fixed samples reacted strongly with the monoclonal antibodies but some variation in the percentages of intact chondrocytes positive by immunofluorescence was noted. Under culturing conditions which induced de-differentiation, epitopes recognized by HuMC1, HuMC3 and HuMC4 disappeared with time in culture. In contrast, reactivities to HuMC2 and HuMC5 either persisted or increased as the culture became more fibroblastic and these antibodies bound to antigens on human fibroblast cell lines. HuMC1, HuMC3 and HuMC4 reacted with purified adult and fetal proteoglycan. HuMC2 and HuMC5 exhibited only slight or no reactivity to either proteoglycan. All five monoclonal antibodies reacted with chondrocytes in frozen articular cartilage but HuMC2 and HuMC5 failed to react to chondrocytes in paraffin-embedded cartilage sections. Only HuMC1, HuMC3 and HuMC4 recognized matrix components in both frozen and paraffin-embedded cartilage. When tested against 29 different non-cartilaginous tissues, each of the monoclonal antibodies had distinctive reactivity patterns, suggesting that each reacted to different epitopes. HuMC3 reacted with neurons in the cerebral cortex and cerebellum, indicating that it may recognize epitopes shared on the S-100 protein. HuMC1 showed the greatest specificity for chondrosarcomas. These antibodies are useful for identifying differentiated chondrocytes, providing information on the distribution of chondrocyte antigens shared by other human tissue, assessing the extent of chondrocyte heterogeneity in a population and aiding in the classification of chondrosarcomas.

Enhanced chondrocyte destruction by lymphokine-activated killer cells. Possible role in rheumatoid arthritis.

OBJECTIVE: The lysis of chondrocytes, the parenchymal cells of cartilage, by lymphocytes may provide a potent mechanism by which the immune system participates in sustaining joint damage in rheumatoid arthritis (RA). We studied the capability of lymphocytes from healthy individuals and patients with arthritis to lyse chondrocytes. METHODS: Peripheral blood mononuclear cells (PMBC) were tested for their ability to lyse chondrocytes in a 51Cr-release assay. Enhancement of the chondrolytic activity was determined by preincubating the cells with T cell growth factor (TCGF) or recombinant interleukin-2 (rIL-2) before cytotoxic testing. RESULTS: PBMC from healthy individuals possessed a low ability to lyse chondrocytes, whereas cells from the synovial fluid of patients with RA displayed higher chondrolytic activity. In RA, modulating factors must come into play because not all synovial fluid sample cells showed high chondrolytic activity and cells from synovial tissue had little or no lytic action on chondrocytes. Chondrolytic activities of cells from all sources, including PBMC from healthy subjects and patients with arthritis and cells isolated from synovial fluid or from the synovial tissue of RA patients, were greatly increased by incubating the cells with TCGF or rIL-2. In contrast, treatment of chondrocytes with interferon-gamma, which enhances major histocompatibility complex gene expression, decreased the susceptibility of chondrocytes to lysis. CONCLUSION: These observations suggest a mechanism for joint damage in which the destruction of chondrocytes by lymphocytes is controlled by cytokines released during the inflammatory process in arthritic diseases.

Treatment of sleep apnea with prosthetic mandibular advancement (PMA).

Nine males with sleep apnea DOES syndrome and three males with sleep apnea DIMS syndrome were treated with prosthetic mandibular advancement (PMA). The method uses a prosthesis, which is designed to advance the mandible 3-5 mm to prevent upper airway occlusion during sleep. The apnea index in the obstructive-type apnea and the percentage of time spent in obstructive apnea decreased significantly with PMA. Although the apnea index showed merely a tendency to decrease in central apnea (p < 0.1), the percentage of time spent in central apnea decreased significantly with PMA. A marked improvement in sleep structures was observed with PMA; a significant increase was seen in total sleep time, percent slow wave sleep (SWS) and percent rapid eye movement (REM) sleep, and the time spent in intra-sleep awakening decreased remarkably. PMA had excellent effects on snoring, and daytime hypersomnolence was reduced in almost all patients. Moreover, a survey on the therapeutic effects of PMA on sleep apnea syndrome and problems associated with wearing PMA was performed with a questionnaire for the sample of nine DOES patients and an additional 22 patients who were treated over a long time. The therapeutic effects could be maintained without any problems in about 2/3 of these patients. The therapeutic mechanisms of PMA in its reduction of both obstructive and central apnea are discussed.

Reaction of antibody to mycobacterial 65 kDa heat-shock protein with human chondrocytes.

We report on the reactivities of four monoclonal antibodies generated against mycobacterial proteins to human chondrocytes, cells in cartilage which may be subject to immune attack in rheumatoid arthritis. Only one of the monoclonal antibodies, ML30, which had been shown previously to react with a human homologue to heat-shock protein (hsp), reacted strongly to chondrocytes. By immunocytochemical methods using fixed chondrocytes, ML30 reacted to cytoplasmic constituents in a granular pattern. There were no marked qualitative differences in the staining intensities and patterns of chondrocytes kept at ambient temperatures and those subjected to 42 degrees C heat treatment. No significant staining was observed with normal peripheral blood mononuclear cells. By indirect immunofluorescence, the distribution of ML30 reactive constituents was very low on the cell surface. Reactivities of each of the monoclonal antibodies were tested on frozen sections of cartilage. Significant reactivity was found only with ML30, and the staining was only associated with chondrocytes, not with the cartilage matrix surrounding the cells. These findings may have significance in view of the possibility that an hsp homologue may be a target for inciting or perpetuating the autoimmune process in rheumatoid arthritis.

Plasmodium falciparum: gene structure and hydropathy profile of the major merozoite surface antigen (gp195) of the Uganda-Palo Alto isolate.

The gene encoding the 195,000-Da major merozoite surface antigen (gp195) of the FUP (Uganda-Palo Alto) isolate of Plasmodium falciparum, a strain widely used for monkey vaccination experiments, has been cloned and sequenced. The translated amino acid sequence of the FUP gp195 protein is closely related to the sequences of corresponding proteins of the CAMP (Malaysia) and MAD-20 (Papua New Guinea) isolates and more distantly related to those of the Wellcome (West Africa) and K1 (Thailand) isolates, supporting the proposed allelic dimorphism of gp195 within the parasite population. The prevalence of dimorphic sequences within the gp195 protein suggests that many gp195 epitopes would be group-specific. Despite the extensive differences in amino acid sequence between gp195 proteins of these two groups, the hydropathy profiles of proteins representative of both groups are very similar. The conservation of overall secondary structure shown by the hydropathy profile comparison indicates that gp195 proteins of the various P. falciparum isolates are functionally equivalent. This information on the primary structure of the FUP gp195 protein will enable us to evaluate the possible roles of conserved, group-specific and variable epitopes in immunity to the blood stage of the malaria parasite.

Merozoite surface coat precursor protein completely protects Aotus monkeys against Plasmodium falciparum malaria.

Groups of Aotus (owl) monkeys were immunized with either the Plasmodium falciparum merozoite surface-coat precursor protein and its processing fragments or a complex of high molecular mass rhoptry proteins and challenged with a lethal infection of the homologous P. falciparum Uganda Palo Alto (FUP) strain. No patent parasitemia could be detected on thick blood films of monkeys immunized with the merozoite surface antigens; however, only one of three monkeys immunized with the rhoptry proteins was partially protected, while two required drug therapy. The experiment clearly demonstrates that the merozoite surface-coat precursor protein can completely protect Aotus monkeys against a lethal infection of the human malaria parasite.

Induction of protective immunity to monoclonal-antibody-defined Plasmodium falciparum antigens requires strong adjuvant in Aotus monkeys.

Monoclonal antibodies to the major Plasmodium falciparum merozoite surface coat and rhoptry antigens were produced. A combination of the affinity-purified polypeptides with Freund complete adjuvant which was given three times completely protected an Aotus lemurinus azure (karotype VI) monkey against homologous challenge; however, immunization with the same polypeptides with a muramyl dipeptide derivative [MDP-Lys(L18)] did not protect a second Aotus monkey, even though comparable high antibody titers were induced.

Plasmodium falciparum: comparative analysis of antigens from continuous in vitro cultured and in vivo derived malarial parasites.

A comparison of metabolically labeled proteins from continuous in vitro and in vivo derived Plasmodium falciparum revealed both similarities and differences. Metabolic labeling of synchronized cultures showed that the uptake of label increased as the parasites matured from the ring to the schizont stage in both cultures. Also, in both continuous in vitro and in vivo derived cultures, prominent high-molecular-weight proteins were synthesized during the late developmental stages. However, the continuous in vitro cultured parasites incorporated twice as much of the label at each stage as did the in vivo derived parasites. Immunoprecipitation with serum samples from vaccinated Aotus trivirgatus griseimembra monkeys revealed major differences involving protein antigens that migrated in the molecular weight regions of b (Mr = 152,000), c (Mr = 143,000), j (Mr = 82,700), and n (Mr = 57,400). These antigens were more readily detected in the continuous in vitro cultured schizonts than in the in vivo derived schizonts.

Plasmodium falciparum: protein antigens identified by analysis of serum samples from vaccinated Aotus monkeys.

Serum samples from Aotus trivirgatus subsp. griseimembra monkeys obtained at different stages of a vaccination experiment were analyzed for total antibody titer to Plasmodium falciparum and were used for identifying protective antigens of the human malaria parasite. Total malarial antibody titers were higher in serum samples from protected monkeys (vaccinated with antigen in an adjuvant) than in those from unprotected monkeys (vaccinated with either antigen or adjuvant only). Parasite proteins were labeled with [3H]isoleucine, solubilized with nonionic detergent, and reacted with immune Aotus sera. Immunoprecipitates obtained were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Thirteen protein antigen bands in the molecular weight range 73,000 to 180,000 were resolved. Serum samples obtained from protected Aotus monkeys reacted more intensely with these proteins than samples from unprotected monkeys did. Evidence is presented that the protective antigen is not a single, normally nonimmunogenic, protein that is recognized only in protected monkeys. Rather, the present data indicate that a heightened immune response to multiple proteins correlated with in vivo protection to P. falciparum in Aotus monkeys. This finding may have a significant bearing on strategies for the development of a human P. falciparum vaccine.

Biochemical studies of H-2K antigens from a group of related mutants. I. Identification of a shared mutation in B6-H-2bm5 and B6-H-2bm16.

Structural studies of the H-2 gene products from a group of five closely related but independent C57BL/6H-2 mutant mice were undertaken. Each of the mutants exhibits reciprocal graft rejection with the parent. The group is remarkable, however, because each member of this group can accept skin grafts from any other member. The results of biochemical analysis of the H-2 glycoproteins from two of these related mutants, bm5 and bm16, are presented in this report. Evidence is given that the H-2K molecules from these two mutants are identical to each other based on comparative tryptic peptide mapping profiles with the parent. From partial amino acid sequence analysis, K products of both mutants have at least one common difference from the parental type located at residue number 116. Definitive studies established that in both bm5 and bm16 a tyrosine found in the parent molecule is substituted with a phenylalanine in the mutant. These results show that a biochemical difference between the K products of the two mutants and of the parent can be detected, that the mutants appear to be identical with one another even though they arose independently, and that they differ from the other H-2Kb mutants analyzed.

Biochemical studies of H-2K antigens from a group of related mutants. II. Identification of a shared mutation in B6-H-2bm6, B6.C-H-2bm7, and B6.C-H-2bm9.

In an earlier paper, we presented evidence that two independent mutants of the bg series, B6-H-2bm5 (bm5) and B6-H-2bm16 (bm16) carry identical mutations such that tyrosine at residue number 116 of the H-2Kb molecule from the parent strain C57BL/6Kh is replaced by a phenylalanine in each of the two mutant molecules. In this paper, we demonstrate, using similar techniques, that the independent bg series mutants B6-H-2bm6 (bm6), B6.C-H-2bm7 (bm7), and B6.C-H-2bm9 (bm9), which share biological properties with bm5 and bm16, can be grouped together because they share two identical mutations, one of which is common to bm5 and bm16, a Tyr to Phe interchange at residue number 116. In addition, a second mutation is at residue number 121, where a Cys in the H-2K molecule from B6 is substituted with an Arg in the mutant. Since all of the bg series mutants arose independently and share biological and biochemical characteristics, it is anticipated that study of these mutants could lead to some understanding of the high mutation rate in the Kb molecule.

Studies on the question of conventional immunoglobulin on thymocytes from primitive vertebrates. I. Presence of anti-carbohydrate antibodies in rabbit anti-trout Ig sera.

An unexpected cross-reactivity between trout immunoglobulin (Ig) and keyhole limpet hemocyanin (KLH) was observed. Rabbit antisera to KLH were capable of binding to radioiodinated trout Ig and, conversely, antitrout Ig reacted with KLH. The cross-reactive antibodies were not found in preimmune sera and did not arise because of a common contaminant in the two immunizing preparations. The molecular basis of the cross-reactivity was found to reside in the carbohydrate moieties. Isolated glycopeptides from KLH and trout Ig were efficient inhibitors of the cross-reactivity. Furthermore, L-fucose was capable of inhibiting the cross-reactivity, whereas other monosaccharides tested did not. Absorption of anti-KLH with trout Ig and anti-trout Ig with KLH effectively removed the cross-reactive antibodies and only slightly affected the titer to their respective homologous antigens. Antibodies with specificity for L-fucose were isolated from anti-KLH and anti-trout Ig sera by passage over affinity columns and elution with the monosaccharide.