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PubMed2026

A Functionally Constrained Immune Ecosystem in Microsatellite-stable Colorectal Cancer Resolved by Single-cell and Exome Profiling.

BACKGROUND/AIM: Microsatellite-stable (MSS) colorectal cancer (CRC) generally responds poorly to immune checkpoint blockade, but some MSS tumors are T-cell rich. We examined whether such infiltration reflected effective immunity or functional immune constraint. CASE REPORT: A 77-year-old woman underwent resection of a mismatch repair-proficient (pMMR), MSS, low-mutational-burden CRC with a synchronous adenoma. Whole-exome sequencing of tumor, adenoma and adjacent normal tissue detected no shared high-confidence somatic mutations between tumor and adenoma within the sensitivity of this WES analysis and identified tumor-specific APC, KRAS and TP53 alterations. Tumor single-cell RNA sequencing yielded 7,569 cells, with T-lineage populations comprising 83.5%. Cytotoxic T cells showed cytolytic and dysfunction-associated features, regulatory T cells (Tregs) showed suppressive remodeling, and Th17 cells showed inflammatory/profibrotic programs. CellChat nominated stromal MIF/FN1-CD74/CD44 and extracellular-matrix communication with T-cell compartments. CONCLUSION: This molecular case report shows that T-cell abundance and immune effectiveness can be uncoupled in MSS CRC.

Humans↗
PubMed1982

Circulating immune complexes and complement in the course of multiple sclerosis.

To evaluate the reactions of the humoral immune response in the course of multiple sclerosis (MS), 21 patients were examined approximately 10 times at regular intervals for 1 year. The study included concomitant investigations of the total neurological deficit (TND) and laboratory analyses of the complement profile, detection of circulating immune complexes (CIC) and heterophilic antibodies (HPA). CIC were found in 58.7% of the investigations, often with simultaneous activation of C4 and C3. The mean values of the complement levels of the MS patients, however, did not differ from the values in a normal population. The consecutive investigations demonstrated a negative regression of TND on C4 from one month previously, a concomitant negative regression of TND on C3 and a positive regression of CIC from one month subsequently on TND. No significant relation between the clinical type of the disease and the occurrence of CIC was demonstrated, but the occurrence of attacks seemed to correlate with development of HPA.

Adult↗
PubMed2002

A gene-expression program reflecting the innate immune response of cultured intestinal epithelial cells to infection by Listeria monocytogenes.

BACKGROUND: Listeria monocytogenes is a Gram-positive, facultative, intracellular bacterial pathogen found in soil, which occasionally causes serious food-borne disease in humans. The outcome of an infection is dependent on the state of the infected individual's immune system, neutrophils being key players in clearing the microorganism from the body. The first line of host defense, however, is the intestinal epithelium. RESULTS: We have examined the transcriptional response of cultured human intestinal epithelial cells to infection by L. monocytogenes, which replicates in the host cell cytoplasm and spreads from cell to cell using a form of actin-based motility. We found that the predominant host response to infection was mediated by NFkappaB. To determine whether any host responses were due to recognition of specific virulence factors during infection, we also examined the transcriptional response to two bacterial mutants; actA which is defective in actin-based motility, and prfA, which is defective in the expression of all L. monocytogenes virulence genes. Remarkably, we found no detectable difference in the host transcriptional response to the wild-type and mutant bacteria. CONCLUSIONS: These results suggest that cultured intestinal epithelial cells are capable of mounting and recruiting a powerful innate immune response to L. monocytogenes infection. Our results imply that L. monocytogenes is not specifically detected in the host cytoplasm of Caco-2 cells by intracellular signals. This suggests that entry of bacteria is mediated in the host cell post-translationally, and that these bacteria seek the cytosol not only for the nutrient-rich environment, but also for protection from detection by the immune system.

Bacterial Proteins↗
PubMed2006

Analysis of bacteria-challenged wild silkmoth, Antheraea mylitta (lepidoptera) transcriptome reveals potential immune genes.

BACKGROUND: In the recent years a strong resemblance has been observed between the insect immune system and the mammalian innate immune mechanisms suggesting their common origin. Among the insects, only the dipterans (Drosophila and various mosquito species) have been widely investigated for their immune responses towards diverse pathogens. In the present study we constructed and analysed the immune transcriptome of the lepidopteran Antheraea mylitta, an economically important Indian tasar silkmoth with a view to unravel the potential immune-related genes and pathways. RESULTS: An expressed sequence tag (EST) library was constructed from mRNA obtained from fat bodies of A. mylitta larvae that had been challenged by infection with Escherichia coli cells. We identified 719 unique ESTs from a total of 1412 sequences so generated. A third of the transcriptome showed similarity with previously characterized immune-related genes that included both the known and putative immune genes. Of the four putative novel defence proteins (DFPs) annotated by PSI-BLAST three showed similarity to extracellular matrix proteins from vertebrates implicated in innate immunity, while the fourth was similar to, yet distinct from, the anti-microbial protein cecropin. Finally, we analysed the expression profiles of 15 potential immune-related genes, and the majority of them were induced more prominently with E. coli compared to Micrococcus luteus. We also identified several unknown proteins, some of which could have probable immune-related functions based on the results of the ProDom analysis. CONCLUSION: The present study has identified many potential immune-related genes in A. mylitta some of which are vertebrate homologues and others are hitherto unreported putative defence proteins. Several genes were present as members of gene families, as has also been observed in other insect species.

Amino Acid Sequence↗
PubMed2004

S100 and cytokine expression in caries.

The molecular immune response of the pulpal tissue during chronic carious infection is poorly characterized. Our objective was to examine the expression of potential molecular mediators of pulpal inflammation, correlate their levels with disease severity, and determine the cellular localization of key molecules. Results indicated that there was significantly increased transcriptional activity in carious compared to healthy pulp, and the increase correlated positively with disease severity. Semiquantitative reverse transcriptase PCR analysis in 10 carious and 10 healthy pulpal tissue samples of the S100 family members S100A8, S100A9, S100A10, S100A12, and S100A13; the cytokines tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-8, IL-6, and epithelial cell-derived neutrophil attractant 78 (ENA-78); and the structural protein collagen-1alpha indicated that all genes tested, with the exception of S100A10, were more abundantly expressed in carious teeth. In addition, we found that the closer the carious lesion front was to the pulpal chamber the higher the expression was for all genes except S100A10. Multiple-regression analysis identified a significant positive correlation between the expression levels of S100A8 and IL-1beta, ENA-78, and IL-6 and between collagen-1alpha and S100A8, TNF-alpha, IL-1beta, IL-8, IL-6, and ENA-78. Immunohistochemical studies in carious pulpal tissue indicated that S100A8 and the S100A8/S100A9 complex were predominantly expressed by infiltrating neutrophils. Gene expression analyses in immune system cells supported these findings and indicated that bacterial activation of neutrophils caused upregulation of S100A8, S100A9, and S100A13. This study highlights the complex nature of the molecular immune response that occurs during carious infection.

Adult↗
PubMed2001

Hormonal response of female goats to active immunization against a recombinant human inhibin alpha-subunit, and establishment of an enzyme-linked immunosorbent assay for caprine follicle-stimulating hormone.

The effect of selective immunosuppression of endogenous inhibin in goats on FSH, LH, progesterone and estradiol-17beta profiles was studied during the breeding and nonbreeding seasons. Eighteen adult female Boer goats were immunized against the recombinant human inhibin alpha-subunit (hINH-alpha). With the exception of estradiol, which was determined by radio-immunoassay (RIA), all plasma hormone concentrations were determined by ELISA. The ELISA for FSH presented in this paper was established in the authors' laboratory, based on an existing RIA. Mean basal concentrations of FSH were not affected by immunosuppression of endogenous inhibin, nor was there a difference in the amplitude of the pre-ovulatory FSH surge. Immunization against inhibin appears to eliminate the slight secondary rise of FSH occurring 12-20 h after the major surge associated with ovulation. The LH profiles of the immunized goats were characterized by lower basal concentrations both before and after the pre-ovulatory LH surge which itself was reduced by 50% in immunized does. By contrast, concentrations of circulating estradiol were significantly elevated after inhibin-immunization. Progesterone profiles were not affected. Extending immunization into the anoestrous season by a booster injection of hINH-alpha, implicating oestrus induction with a progestagen and eCG, produced no discernible differences in FSH and LH profiles in comparison with nonimmunized control goats. The findings suggest that in goats, paracrine factors may play a more significant role in controlling follicular activity than a feedback mechanism acting via the pituitary.

Animals↗
PubMed1991

Effects of active immunization against somatostatin (SRIF) and/or injections of growth hormone-releasing factor (GRF) during gestation on hormonal and metabolic profiles in sows.

The hormonal responses of gestating sows to immunization against somatostatin conjugated to bovine serum albumin (SRIF-IMM) and/or injections of growth hormone-releasing factor (GRF) were studied with thirty-eight second parity sows. Immunization against bovine serum albumin (BSA-IMM) was used as control. First immunizations were done on day 30 and boosters were given on days 44, 58, 72, 86 and 100 of gestation. Injections of GRF (9 mg of GRF (1-29)NH2 per injection) or saline were given at 0800, 1400 and 2000 hr daily from day 90 of gestation until parturition. Mean body weights of sows at 85 and 110 d of gestation were 196.3 and 210.5 kg, respectively (SE = 0.8). Jugular blood samples were collected from 0740 hr to 1100 hr at 20 min intervals on days 90, 101 and 112 of gestation. On day 112, additional samples were collected from 1340 hr to 1700 hr and from 2140 hr to 2300 hr. At 112 d of gestation, antibody titers against SRIF (% binding, 1:150 dilution) were higher (P less than 0.01) for SRIF-IMM (13.5%) vs BSA-IMM (0.95%) sows. There was no effect of SRIF-IMM nor was there a GRF by SRIF-IMM interaction on any variable measured (P greater than 0.05). Injections of GRF increased (P less than 0.01) the area under the curve (AUC) for growth hormone (GH; 305 vs 1623 ng/min/ml). The increase was greater as days of injection increased (P less than 0.05). Administration of GRF did not affect prolactin (Prl) AUC (P greater than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1977

Isoelectric focus analysis of rat anti-phosphocholine antibodies.

Anti-phosphocholine (PC) antibodies in sera from four strains of rats were examined before and afterimmunization with either Streptococcus pneumoniae R36A, which contains PC as a cell wall component, or with PC-coupled keyhole limpet hemocyanin (PC-KLH). PC-specific protein was purified from pooled immune sera and shown by a combination of isoelectric focus (IEF) in acrylamide and crossed immunoelectrophoresis, as well as by molecular weight determination in NaDodSO4-acrylamide, to be immunoglobulin. An additional, small molecular weight, nonimmunoglobulin protein (pI = 7.1-7.3) was present in sera from normal and germ-free rats which had the ability to bind the C-carbohydrate of S. pneumoniae R36A, but without specificity for PC. The IEF profile of normal and immune sera showed marked sharing of bands of anti-PC antibody between individual rats as well as between strains. In addition, other anti-PC antibodies which focused between pH 8.5 and 9.5 were less regularly shared. The uniformity of IEF profile of the bulk of anti-PC antibodies in rats is most consistent with their being the products of germ line genes.

Animals↗
PubMed1988

Alloantibodies, autoantibodies, and immune complexes in patients with lung cancer.

The sera of patients with lung cancer, nonmalignant lung disease, and blood donors were subjected to various immunologic assays. Nine assays, based on immunoradiometric (IRMA) and immunoenzymatic (ELISA) principles, included 3 types of fetal cell antibodies, 2 established lung cancer cell antibodies, anti-DNA, anti-IgG autoantibodies, and immune complex assays based on C1q binding and anti-C3 activity. Antitumor cell antibody level was significantly lower in patients with lung cancer compared to blood donors. In the remaining 7 assays, the lung cancer patients tended towards higher median values compared to both control patients and blood donors, but without statistical significance, with the exception of anti-DNA antibodies. Statistical analysis of all 9 assays taken together has shown significant differences between the 3 groups. When only 5 assays were used to assess 3 types of fetal cell antibodies, anti-DNA antibodies, and immune complexes by means of ELISA anti-C3, the margins between groups increased. A range of values for the selected assays was established that may discriminate 70% of tested individuals of the 3 groups. These results suggest the existence of a characteristic profile of deranged humoral immunity in lung cancer patients.

Adenocarcinoma↗
PubMed1999

Immune determinants of organism and outcome in febrile hospitalized Thai patients with bloodstream infections.

Opportunistic infections (OI) and the human immunodeficiency virus (HIV) cause significant morbidity and mortality in developing countries. Immune cell and cytokine profiles may be related to the type and course of OI and to the OI-HIV interaction. Examining cell-specific cytokine production ex vivo has only recently become feasible. In Thailand, 53 febrile, hospitalized adults were enrolled in a study of the immune correlates of bloodstream infections (BSI). On site, blood cells were stimulated ex vivo. Cell-surface antigens and eight intracellular cytokines were subsequently analyzed using flow cytometry to determine associations with mortality and the organism causing the BSI. By logistic regression analysis, the percentage of CD3(+) CD16/56(+) cells making tumor necrosis factor alpha (TNF-alpha) (P = 0.033) and the percentage of CD3(-) CD16/56(+) cells (NK) (P = 0.032) were related to HIV positivity. Lymph node enlargement with HIV infection and the percentage of CD3(+) CD16/56(+) making TNF-alpha were predictive of death. A lower percentage of CD3(+) CD8(+) lymphocytes making interleukin-8 (IL-8) (P = 0.005), fewer monocytes expressing CD14 (P = 0.009), and the percentage of CD3(+) CD8(+) cells producing gamma interferon (P = 0. 011) were associated with blood culture positivity and the causative organism. For every one point decrease in the percentage of CD3(+) CD8(+) cells making IL-8, the likelihood of a positive culture increased 23%; for every one point decrease in the percentage of monocytes expressing CD14, the likelihood of a positive culture increased by 5%. Only a few immune cell types and three of their related cytokines were significantly associated with HIV disease outcome or the BSI organism. These cell types did not include CD3(+) CD8(-) cells (a surrogate for CD4(+) cells), nor did they involve cytokines associated with a type I to type II cytokine shift, which might occur with advancing HIV infection. These associations support the premise that CD8(+) and CD16/56(+) lymphocytes play significant roles in HIV and type I infections.

AIDS-Related Opportunistic Infections↗
PubMed1996

IL-15 augments CD8+ T cell-mediated immunity against Toxoplasma gondii infection in mice.

Cytokines of the Th1 profile are important mediators of protective host immunity against Toxoplasma gondii infection in mice. In this study we describe the effect of the recently identified cytokine, IL-15, on prevention of murine infection with T. gondii. Administration of exogenous rIL-15 with soluble Toxoplasma lysate Ag (TLA) provides complete protection against a lethal parasite challenge, whereas treatment with either rIL-15 or TLA alone is not protective. Following immunization with TLA/rIL-15, there is a significant proliferation of splenocytes expressing the CD8+ phenotype in response to TLA. A significant rise in the level of serum IFN gamma was observed in vaccinated mice. Adoptive transfer of CD8+ T cells, but not CD4+ T cells, from TLA/rIL-15-vaccinated mice protects naive mice from a lethal parasite challenge. These CD8+ T cells exhibit enhanced CTL activity against target macrophages infected with T. gondii. Mice that have been immunized are protected against lethal parasite challenge for at least 1 mo postvaccination. These observations demonstrate that TLA when administered with exogenous rIL-15 generates toxoplasmacidal Ag-specific CD8+ T cells. These T cells proliferate upon exposure to parasite Ag, exhibit long term memory CTL against infected target cells, and may be involved in host immune memory to this parasite.

Adjuvants, Immunologic↗
PubMed2002

Open system gene expression profiling and identification of novel genes for targeted vaccine delivery.

Ongoing development of vaccines is limited by the techniques used to identify routes to immunization. Using open system gene expression profiling, researchers can now directly identify receptors used by M-cells in the transport of antigens to the mucosal immune system. These receptors will be ideal for targeted delivery of new synthetic vaccines. In addition, gene expression profiling will provide important new information on the biology of the M-cell and mucosal immune mechanisms.

Gene Expression Profiling↗
PubMed2006

Importance of the host specificity in the selection of probiotic bacteria.

The gastrointestinal tract is a complex and dynamic ecosystem. Commensal microorganisms (C), which proliferate in the intestine from birth, are crucial for gut homeostasis while non commensal (NC) microorganisms are transient and enter the organism from the environment and foods. We studied comparatively the influence of oral administration of C and NC Lactobacillus fermentum and Lactobacilus acidophilus on the gut-associated lymphoid tissue (GALT) of conventional mice. To determine the importance of the selection of probiotic host-specificity bacteria with immunomodulating capacity, we examined the interaction with the gut by transmission electron microscopy and FITC-labelled bacteria. We compared the immunomodulation capacities of C and NC strains by studying the number of IgA secreting cells and cytokine profile. No differences were found in the number of IgA+ cells; however, the pattern of cytokine response to C and NC bacteria was different. With regard to proinflammatory cytokine (IFNgamma and TNFalpha), we found that TNFalpha was mainly produced by NC bacteria, while C bacteria were able to elicit mainly IFNgamma. The regulatory cytokines (IL-10 and IL-4) were induced with different patterns for both C and NC strains. No differences in the pathway of internalization to the gut between C and NC were found. In summary, we determined that C and NC bacteria interact with the intestine in the same way; both C and NC bacteria were able to reinforce the surveillance of the gut mucosal immune system. The cytokine profile showed that C bacteria would be involved in the regulation of intestinal homeostasis rather than in the immune activation as the NC bacteria.

Administration, Oral↗
PubMed2005

Emergence of immune escape variant of mammary tumors that has distinct proteomic profile and a reduced ability to induce "danger signals".

Breast tumors are shaped, in part, by a process termed immunoediting which selects for immunologically evasive phenotypes. In the present study we used the rat neu-transgenic mouse model of breast cancer and its congenic non-transgenic parental strain, FVB, to explore the phenotype of tumors that emerge in the presence of an immune response directed against the neu antigen. When inoculated into parental FVB mice, a neu-overexpressing mouse mammary carcinoma (MMC) cell line isolated from spontaneous breast tumors of the FVB neu (FVBN202) transgenic mouse, elicited a neu-specific immune response resulting in a tumor rejection because of the presence of the rat neu antigen. However, a neu negative variant (ANV) of MMC arose after a long latency in spite of the neu-specific immune response. We show that compared to MMC, ANV tumor cells have a significantly reduced ability to secrete pro-inflammatory cytokines and the CCL5 chemokine, to express immunostimulatory chaperones, and they have a distinct expression of proteins involved in cell motility, and metabolic and signal transduction pathways. These studies suggest that tumor escape through immunoediting can not be explained by the loss of a single tumor antigen, but rather by a selection process of a tumor variant that has a reduced ability to induce "danger signals" together with up-regulation of proteins involved in the tumor survival. Based on these findings, we propose to target novel antigens over-expressed in the escape variant of breast tumors to treat primary tumor and to prevent tumor relapse.

Animals↗
PubMed1998

Oral administration of schistosome egg antigens and insulin B-chain generates and enhances Th2-type responses in NOD mice.

In murine Schistosoma mansoni, parenteral administration of parasite eggs or saline-soluble egg antigens (SEA), generates Th2 T-cell responses to both schistosome-specific and unrelated third-party antigens. Oral administration of insulin to NOD mice suppresses or delays the onset of diabetes by skewing the response toward CD4+ Th2 cells and TGF-beta producing cells. From these two independent sets of observations, we initiated the present study to determine if oral administration of SEA would stimulate Th2-type cytokine responses when mice were fed SEA alone or in tandem with insulin B-chain. Our results show that feeding NOD mice with either insulin B-chain or SEA alone significantly inhibits proliferation to the immunizing antigen. When cytokine profiles were examined, feeding led to a predominance of IL-10 and TGF-beta production. Furthermore, feeding SEA in combination with insulin B-chain augmented the level of IL-10 production to insulin. T-cell lines established from SEA-fed and -immunized mice secreted IL-4 and IL-10 cytokines whereas the T-cell lines from control-fed mice immunized with SEA secreted predominantly IL-2 and IFN-gamma. These results demonstrate that orally administered insulin can induce regulatory T-cells secreting IL-4, IL-10, and TGF-beta and that Th2 responses to oral insulin could be augmented in a synergistic way by feeding SEA and insulin B-chain together.

Administration, Oral↗
PubMed2006

Increased alpha2,6-sialylation of surface proteins on tolerogenic, immature dendritic cells and regulatory T cells.

OBJECTIVE: Surface protein glycosylation of lymphocytes plays a key role in development, maturation, and immune regulation. Sialic acid most often is the terminal carbohydrate in these posttranslational modifications. Receptors for sialic acids are expressed on lymphocytes and can generate an inhibitory signal. This study compared the sialic acid expression pattern of tolerogenic cells and effector cells. METHODS: Gene expression profiles of immature and mature monocyte-derived dendritic cells were compared using cDNA array technology. We analyzed the cell-surface protein sialylation of dendritic cells and different T cell subpopulations by flow cytometry using plant lectins. RESULTS: Monocyte-derived dendritic cells showed a separation according to alpha2,6-linked sialic acid density. Tolerogenic, immature DC showed a higher alpha2,6-linked sialic acid, which was drastically downregulated after maturation of DC with proinflammatory cytokines. This differential expression of alpha2,6-linked sialic acid was reflected by transcriptional regulation of specific glycosyl transferases during DC maturation shown by cDNA array analysis. Furthermore, CD4(+) T cells significantly upregulated alpha2,6-linked sialic acid density, whereas alpha2,3-linked sialic acid density remained largely unchanged after stimulation. Isolated CD4(+)CD25(+) T cells showed a population with high density of alpha2,6-linked sialic acid and a population with low expression. The density of this particular carbohydrate was further increased during culture conditions expanding inhibitory T cells. CONCLUSION: Surface proteins on tolerogenic, immature dendritic cells and regulatory T cells are highly alpha2,6-sialylated, suggesting a glycan motif of tolerogenic cells which might serve as ligand for inhibitory siglecs on the surface of effector cells.

Carbohydrate Sequence↗
PubMed2000

Shedding of lipopolysaccharide and 200-kDa surface antigen during the in vitro growth of virulent Ara- and avirulent Ara+ Burkholderia pseudomallei.

Non-virulent Ara+ B. pseudomallei environmental isolates differ from virulent Ara- clinical isolates by their ability to assimilate L-arabinose and the absence of a 200 kDa antigen on their surface. The latter, present only on the Ara- isolates from either clinical or environmental origin, was recently demonstrated by its immunoreactivity with monoclonal antibody (MAb) 5F8. We recently demonstrated that lipopolysaccharide (LPS) from both biotypes were indistinguishable from one another with regard to SDS-PAGE profiles and immunoreactivities with immune sera. In this study, the shedding of LPS and 200-kDa antigen into the culture medium during the in vitro growth of Ara- was compared with that of its Ara+ counterpart, using MAb-based sandwich ELISAs. The results showed that the LPS shedding profiles from the two biotypes were similar to one another. This was in contrast to the situation with the 5F8-reactive antigen. The culture fluid of all Ara- isolates and none of the Ara+ isolates were found to react strongly with the MAb 5F8 during the early log phase of growth. However, during the late stationary phase, a trace amount of the 5F8-reactive material could also be detected in the culture fluid of the Ara+ isolates.

Antibodies, Monoclonal↗
PubMed1996

T lymphocyte mediated protection against facultative intracellular bacteria.

Acquired immunity against intracellular bacteria is T cell dependent. T cells play a major role in protection against intracellular bacteria, but bacterial antigens recognized by T cells have been studied less extensively than bacterial antigens recognized by B cells. Using T lymphocytes from animals immunized against Brucella abortus, we have screened a bacterial genomic library for genes encoding antigens recognized by T cells. Lymphocytes that proliferated to B. abortus proteins were characterized for phenotype and cytokine activity. Bovine and murine lymphocytes recognized common bacterial antigens and possessed similar cytokine profiles, suggesting an analogous immune response in these two animal species. In vivo protection afforded by a particular cell type is dependent on the bacterial antigens presented and mechanisms of antigen presentation. MHC class I and class II gene knockout animals infected with B. abortus have demonstrated that protection to B. abortus is especially dependent on CD8+ T cells. Knowing the cells required for protection, vaccines can be designed to elicit the protective subset of lymphocytes. Currently, we are testing several recombinant B. abortus proteins using different immunization strategies. Finally, bacterial genes activated following intracellular phagocytosis are being examined using a novel, reporter system adapted to B. abortus.

Animals↗