The major gangliosides of human peripheral blood monocytes/macrophages: absence of ganglio series structures.

Sialoglycosphingolipids (gangliosides) are membrane components of eukaryotic cells that modulate cell signal transduction events. Discrepancies exist in the published descriptions of the gangliosides present in the human peripheral monocyte/macrophage. Macrophages were isolated from healthy human volunteers by two different methods. Their ganglioside fractions were isolated and examined by 2D thin-layer mobility, enzymatic susceptibility, and mass spectral-collision induced dissociation-mass spectral analyses. Thin-layer ganglioside chromatographic patterns displayed four major doublets and were similar for monocytes/macrophages isolated by either apheresis/elutriation or density gradient centrifugation. All gangliosides were resistant to beta-galactosidase but sensitive to Clostridium perfringens sialidase, indicating the absence of terminal galactose residues and sialidase-resistant sialic acid moieties. Mass spectra indicated only three major sets of glycolipid components with mass heterogeneity in the ceramide portion of each set. In all the gangliosides, the ceramide moiety contained only C18 sphingosine with the heterogeneity produced by the presence of C16 or C24 fatty acid. One doublet was resistant to Newcastle disease virus sialidase, indicating the presence of an alpha(2-6)-linked sialic acid residue with the same mass as another doublet. All data was consistent with the following structures as the major gangliosides of human peripheral monocyte/macrophages: II(3)NeuAcLacCer (sialolactosyl ceramide, GM3), IV(3)- and IV(6)NeuAcnLcOse(4)Cer (sialoparagloboside, nLM1), and IV(3)NeuAcnLcOse(6)Cer (a sialohexosylceramide).

Differences in splenic B-lymphocyte ganglioside expression and accessibility in normal and endotoxin-hyporesponsive mice.

Endotoxin-responsive (C3H/HeN) and -hyporesponsive (C3H/HeJ) murine B lymphocytes purified by adherence to anti-immunoglobulin ("antibody panning") possess identical gangliosides but different ganglioside surface accessibilities. We investigated the distribution and surface accessibility of gangliosides of B lymphocytes purified by adherence to plastic ("plastic panning") or by subtraction of non-B-lymphocyte components. As with antibody panning, there were no entirely new or absent gangliosides in plastic-panned or subtraction-purified B lymphocytes of each strain. However, striking changes in relative expression of five gangliosides were detected with each purification protocol. Moreover, five gangliosides of antibody-panned and plastic-panned B lymphocytes but only two gangliosides of subtraction-purified B lymphocytes were inaccessible to surface labeling. Unlike the situation for antibody-panned B lymphocytes, no interstrain (HeN vs. HeJ) surface accessibility differences existed in gangliosides of plastic-panned or subtraction-purified cells. Exposure of subtraction-purified B lymphocytes to anti-immunoglobulin failed to elicit changes in ganglioside expression. Murine B lymphocytes have distinct protocol-dependent differences in glycolipid phenotype which likely denote individual subpopulations.

Suppression of a sialyltransferase by antisense DNA reduces invasiveness of human colon cancer cells in vitro.

Transfer of terminal alpha 2,6-linked sialic acids to N-glycans is catalyzed by beta-galactoside alpha 2,6-sialyltransferase (ST6Gal I). Expression of ST6Gal I and its products is reportedly increased in colon cancers. To investigate directly the functional effects of ST6Gal I expression, human colon cancer (HT29) cells were transfected with specific antisense DNA. ST6Gal I mRNA and protein were virtually undetectable in six strains of transfected HT29 cells. ST6Gal activity was reduced to 14% of control (P<0.005) in transfected cells. Expression of terminal alpha 2,6- and alpha 2,3-linked sialic acids, and unmasked N-acetyllactosamine oligosaccharides, respectively, was assessed using flow cytometry and fluoresceinated Sambucus nigra, Maackia amurensis and Erythrina cristagalli lectins. Results indicated a major reduction in expression of alpha 2,6-linked sialic acids and counterbalancing increase in unmasked N-acetyllactosamines in antisense DNA-transfected cells, without altered expression of alpha 2,3-linked sialic acids or ganglioside profiles. The ability of transfected cells to form colonies in soft agar and to invade extracellular matrix material (Matrigel), respectively, in vitro was reduced by approx. 98% (P<0.0001) and more than 3-fold (P<0.005) compared to parental HT29 cells. These results indicate that N-glycans bearing terminal alpha 2,6-linked sialic acids may enhance the invasive potential of colon cancer cells.

Idiosyncratic rifabutin-induced leukopenia and SIADH: case report and review.

Rifabutin is increasingly critical for treatment of atypical mycobacterial infections. One of its serious adverse effects is leukopenia. When encountering rifabutin-induced leukopenia, clinicians are faced with the dilemma of whether to lower the dosage of rifabutin or discontinue it because existing literature does not indicate whether rifabutin-induced leukopenia is dose related or idiosyncratic. We report the first established case of idiosyncratic rifabutin-induced leukopenia in an immunocompetent man treated for pulmonary Mycobacterium avium complex infection. The patient also developed rifabutin-induced syndrome of inappropriate antidiuretic hormone (SIADH), which also has not been previously reported.

Gangliosides of monocyte-derived macrophages of adults with advanced HIV infection show reduced surface accessibility.

Gangliosides of macrophages are potent immunoregulatory molecules. A monoclonal antibody directed at human macrophage gangliosides (25F4) inhibits macrophage migration with relative specificity. Recent reports suggested that greater expression of G(M1) in mononuclear cells accompanies advanced HIV infection, although others failed to demonstrate any differences in vitro. We purified gangliosides from blood monocyte-derived macrophages obtained from HIV-infected adults. Densitometric analysis of chromatograms demonstrated no differences in relative quantities of any macrophage gangliosides among all HIV-positive and -negative donors. Antibody 25F4 showed equivalent ELISA reactivity with purified macrophage gangliosides of HIV-positive and -negative donors. However, intact macrophages of HIV donors with CD4+ cell counts <200/mm3 showed impaired immunofluorescent surface expression of the 25F4 epitope and concomitant loss of migration inhibitory responsiveness. Thus, although relative content is unchanged, macrophage gangliosides become surface-inaccessible in adults with advanced HIV infection. Our data provide further evidence that dysregulation of glycosphingolipid metabolism in HIV-1 infection contributes to immune dysfunction.

Human thyroid fibroblasts exhibit a distinctive phenotype in culture: characteristic ganglioside profile and functional CD40 expression.

Fibroblasts from different regions of the human body exhibit substantial phenotypic diversity, some of which relates to the capacity for cross-talk with cells of the immune system. We examine, for the first time, thyroid fibroblast biology in culture. Thyroid explants were placed in culture, and fibroblasts were outgrown and serially passaged. These fibroblasts take on a morphology in culture resembling cells from other anatomic regions. When treated with PGE2, they assume a stellate morphology similar to that of prostanoid-treated orbital fibroblasts. The ganglioside profile exhibited by these cells is distinct from that observed previously in orbital and dermal fibroblasts. They uniformly express Thy-1, a surface glycoprotein. Messenger RNA encoding CD40, a surface receptor found on bone marrow-derived cells, and CD40 protein were expressed constitutively at low levels. Interferon-gamma (500 U/ml) treatment for 48-72 h resulted in high levels of surface HLA-DR and CD40 display. When CD40 is engaged with CD40 ligand (CD40L), nuclear factor-kappaB binding activity is up-regulated as is interleukin (IL)-6 and IL-8 expression. IL-1beta treatment up-regulates the expression of IL-1alpha, IL-1beta, and PGE2. These observations suggest that thyroid fibroblasts possess the molecular machinery necessary for cross-talk with immunocompetent cells such as lymphocytes and mast cells through the CD40/CD40L complex, as well as through classic cytokine networks, and to participate potentially in the inflammatory response of the thyroid gland.

Specific binding of Haemophilus influenzae to minor gangliosides of human respiratory epithelial cells.

Gangliosides are sialylated glycosphingolipids that serve as receptors for various bacteria. To investigate endogenous gangliosides of human respiratory epithelial cells as potential receptors for Haemophilus influenzae, three strains, including nontypeable H. influenzae (NTHI) 1479, and isogenic fimbriated (f+) and nonfimbriated (f0) H. influenzae type b 770235, were 3H labeled and overlaid on two-dimensional thin-layer chromatography (TLC) plates containing either purified HEp-2 gangliosides or murine brain gangliosides. NTHI 1479 bound exclusively to two distinct minor ganglioside doublets, with mobilities near that of GM1. These minor gangliosides comprised only 14.2 and 9.4% of the total, respectively. NTHI 1479 also bound to a distinct ganglioside of human macrophages whose chromatographic mobilities closely resemble those of one of the NTHI-binding gangliosides of HEp-2 cells. H. influenzae type b 770235 f+ and f0 each bound to a different minor HEp-2 ganglioside doublet, with proportionately weaker affinity for a major ganglioside doublet. Remarkably, none of the three strains bound to any murine brain gangliosides. Moreover, when 80 to 90% of sialic acid residues were enzymatically removed from HEp-2 gangliosides, NTHI 1479 binding was proportionately impaired, compared with untreated controls. Our findings support a role for specific gangliosides of specific cells as receptors for H. influenzae strains. Our findings further demonstrate that individual minor gangliosides possess unique biological properties.

A monoclonal antibody to human macrophage gangliosides inhibits macrophage migration.

Gangliosides have diverse immunoregulatory properties. The gangliosides endogenous to macrophages may have immunoregulatory properties that distinguish them from other gangliosides. Gangliosides have been indirectly implicated in macrophage migration as putative cell surface receptors for migration inhibitory factor (MIF). In this study, a monoclonal antibody to human macrophage gangliosides (antibody 25F4) was developed and characterized. This is the first report of the development of monoclonal antibodies to gangliosides of macrophages of any species. Thin-layer chromatographic immunostaining indicated that antibody 25F4 recognized major gangliosides of human macrophages but did not recognized those previously identified as containing fucose. Immunofluorescent surface labeling of viable human macrophages indicated that antibody 25F4 recognized a surface-accessible epitope, present on all cells, and that this was abolished with lipid depletion of macrophage membranes. This epitope was not present on several human nonmacrophage cells. Finally, human macrophages pretreated with antibody 25F4 demonstrated striking inhibition of migration of an agarose droplet assay, whereas an irrelevant monoclonal antibody or monoclonal antibodies to nonganglioside surface epitopes of human macrophages had no effect on migration. Migration inhibition occurred even though antibody 25F4 was removed from the extracellular milieu and was not due to formation of cellular aggregates. These studies support a role for human macrophage gangliosides in macrophage migration.

Human orbital fibroblasts in culture express ganglioside profiles distinct from those in dermal fibroblasts.

Orbital fibroblasts appear phenotypically distinct from those derived from dermis and other extraorbital anatomical sites. In this study, we examined the profile of gangliosides expressed by orbital and dermal fibroblasts. Gangliosides have a wide range of functions including modulation of transmembrane signal transduction. The aim of this study was to examine the hypothesis that a differential expression of gangliosides by orbital and nonorbital fibroblasts could constitute an important determinant of the immunological properties peculiar to the orbit. Moreover, these differences could provide a molecular basis for the site-specific involvement of the orbit in Graves' ophthalmopathy. Total lipids were extracted from confluent cultures of six different orbital and six dermal fibroblast strains, and purified gangliosides were subjected to two-dimensional thin layer chromatographic analysis. Orbital and dermal fibroblasts contained qualitatively similar ganglioside contents, with two major peaks, one migrating in the mono- and the other in the disialoganglioside regions of the chromatogram. In orbital fibroblasts, the densities of these two peaks were nearly equal, whereas in dermal fibroblasts, the monosialoganglioside peak was 5- to 6-fold greater. Minor ganglioside peaks were resolved and were equally abundant in orbital and dermal fibroblasts. Ganglioside profiles were invariant with respect to treatment of fibroblasts with interferon-gamma. These differences in expression of the two major ganglioside species may be relevant to the peculiarities associated with normal and pathological events in orbital connective tissue.

n-Butyrate mediation of ganglioside expression of human and murine cancer cells demonstrates relative cell specificity.

1. n-Butyrate, a short chain fatty acid produced by colonic fermentation, induces differentiation in human neoplastic cell lines, and reduces expression in vitro of a sialyltransferase that glycosylates N-linked glycoproteins in hepatoblastoma cells. Gangliosides are amphipathic, sialylated glycosphingolipids that undergo profound changes in many transformed cells and may protect neoplastic cells from host immune surveillance. Colonic mucosal cells are exposed to luminal short-chain fatty acid concentrations of up to 80 mmol/l, and there is some evidence that short-chain fatty acids may alter ganglioside expression in colon cancer cells. 2. Because of the importance of gangliosides in cancer pathogenesis, we investigated the effects of n-butyrate on ganglioside expression of colonic (human and murine) and non-colonic cancer cells. 3. Three separate colon cancer cell lines (LS174T, T84 and MCA-38), when butyrate treated, demonstrated striking amplification of specific individual gangliosides. However, the total lipid-bound sialic acid content of gangliosides of butyrate-treated LS174T cells diminished. In contrast to earlier reports, n-butyrate did not mediate expression of all gangliosides and specifically did not mediate expression of GM3. This effect persisted even after removal of butyrate. 4. In contrast, exposure of extracolonic cells to butyrate, including cervical cancer (HeLa) and laryngeal cancer (HEp-2) cell lines in this study and hepatoblastoma cells (Hep G2) in our previous work, caused no detectable changes in ganglioside expression. 5. In conclusion, our results indicate a relative tissue specificity of butyrate-mediated alterations in ganglioside expression that is not universal but is limited to specific gangliosides.

Determinants of differential liver-colonizing potential of variants of the MCA-38 murine colon cancer cell line.

We investigated factors that might contribute to the differing liver tumor colonizing potentials of MCA-38 colonic cancer cell line variants injected into the ileocolic veins of C57Bl/6J mice. Non-colonizing (MCA-38 CD) cells were sensitive to lysis by hepatic natural killer (NK) cells in vitro (51Cr-release assay) and cells with high liver-colonizing potential (MCA-38 LD) were resistant. Following abrogation of NK activity by treatment with anti-asialoGM1, liver-colonizing ability to LD cells but not CD cells was enhanced. MCA-38 CD cells were, however, capable of initial liver colonization after ileocolic vein injection. Differing patterns of membrane sialylation may have contributed to the contrasting hepatic tumorigenicities of LD and CD cells; beta-galactoside alpha 2,6-sialyltransferase mRNA levels and activity were approximately four-fold higher in LD than CD cells and qualitative and quantitative differences existed between their ganglioside profiles. In the MCA-38 model outlined, tumor cell susceptibility or resistance to NK lysis was a relatively unimportant determinant of liver-colonizing potential.

First reported case of Aspergillus granulosus infection in a cardiac transplant patient.

We report a case of disseminated infection with Aspergillus granulosus in a cardiac transplant recipient on immunosuppressive therapy. This is the first reported case in which this organism has been described as a pathogen. This organism bears morphological features different from those of more common Aspergillus species and should be considered a potential pathogen in immunocompromised patients.

n-butyrate reduces the expression of beta-galactoside alpha 2,6-sialyltransferase in Hep G2 cells.

n-Butyrate, a short chain fatty acid that is produced by colonic bacterial fermentation, is detectable in portal blood and induces differentiation in various human neoplastic cell lines. Earlier reports indicated approximately 20-fold induction in vitro by n-butyrate of the sialyltransferase that catalyzes terminal glycosylation of GM3 ganglioside in HeLa and colon cancer cells. We previously isolated a 1.3-kilobase cDNA for a human beta-galactoside alpha 2,6-sialyltransferase, for which N-linked glycoproteins are the acceptors. We report here that treatment of Hep G2 cells with 5 mM n-butyrate for 24 h reduced beta-galactoside alpha 2,6-sialyltransferase mRNA levels by approximately 90%. Reductions in mRNA level were followed by approximately 75 and approximately 90% reductions, respectively, in specific beta-galactoside alpha 2,6-sialyltransferase enzyme activity after treatment for 24 and 36 h with 5 mM n-butyrate. However, in contrast with earlier reports of enhanced ganglioside synthesis in response to n-butyrate treatment, incubation of Hep G2 cells with n-butyrate did not alter the ganglioside pattern as assessed by thin layer chromatography of lipids extracted from treated cells. Nuclear run-on reactions indicated that the rate of transcription of beta-galactoside, alpha 2,6-sialyltransferase was not altered by treatment with 5 mM n-butyrate for 24 h, but the effects of this treatment on cytoplasmic levels of beta-galactoside alpha 2,6-sialyltransferase mRNA were largely negated by co-treatment with actinomycin D or cycloheximide. Therefore, our results show that n-butyrate reduces expression of mature beta-galactoside alpha 2,6-sialyltransferase mRNA by post-transcriptional mechanisms.

Murine peritoneal macrophage gangliosides inhibit lymphocyte proliferation.

Gangliosides have been shown to act as immunoregulatory agents by altering proliferative responses of lymphocytes to both antigens and mitogens. Most early studies have utilized brain gangliosides and have required high concentrations. The role of endogenous gangliosides from macrophages has remained unexplored. In this study, thioglycolate-elicited murine peritoneal macrophage gangliosides were purified and added to cultures of murine lymphocytes. Nanogram amounts caused a profound inhibition of LPS-induced DNA synthesis of splenocytes and of purified B lymphocytes, without demonstrable cellular toxicity. No effect was seen using asialo-GM1. This effect was present across a wide range of lipopolysaccharide (LPS) doses. Nanogram amounts of macrophage gangliosides also inhibited concanavalin A (ConA)-mediated lymphocyte proliferation. Inhibition of LPS-induced mitogenesis was present even if gangliosides were removed from the extracellular environment after 15-60 min of incubation prior to the addition of LPS. This inhibition was reversible with incubation of ganglioside pre-treated lymphocytes in medium containing serum. These inhibitory properties of macrophage gangliosides are distinct from those found in studies using brain gangliosides, and support a potential role for macrophage gangliosides as negative modulators of lymphocyte proliferation.

Altered B-lymphocyte membrane architecture indicated by ganglioside accessibility in C3H/HeJ mice.

We have analyzed both the total ganglioside composition and the surface accessibility of C3H/HeN B lymphocytes and C3H/HeJ B lymphocytes. Seventeen individual resorcinol-positive moieties were visualized by two-dimensional thin-layer chromatography of the purified gangliosides from both strains. Complete homology between strains was seen in the patterns of total gangliosides purified from the endotoxin-responsive and -hyporesponsive strains, with only minor differences in the relative concentrations of four gangliosides. In comparison, only 12 individual gangliosides were accessible to surface labeling following galactose oxidase treatment in these same strains, suggesting that some gangliosides are masked at the cell surface in both strains. However, labeling of the more polar components was greatly reduced in the endotoxin-hyporesponsive (C3H/HeJ) strain, suggesting that these gangliosides have decreased accessibility to galactose oxidase at the cell surface. Therefore, while the total ganglioside compositions of the two strains were nearly equivalent, there were dramatic differences in ganglioside surface accessibility. These findings indicate that an alteration in membrane structure that is associated with the endotoxin hyporesponsiveness observed in C3H/HeJ B lymphocytes exists.

Propionibacterium acnes causes postoperative brain abscesses unassociated with foreign bodies: case reports.

Propionibacterium acnes was isolated in pure culture from brain abscesses that occurred in two patients after intracranial surgery. Although such infections are usually associated with cerebrospinal fluid shunts and other foreign bodies, these cases clearly demonstrate the pathogenicity of this anaerobic diphtheroid in the absence of such. Progression of P. acnes infection in the central nervous system can be insidious. To treat such infections adequately, therapy cannot rely upon some standard antimicrobial agents. Metronidazole, which is useful against most anaerobic organisms, is not effective against P. acnes.

Factors mediating lipopolysaccharide-induced ganglioside expression in murine peritoneal macrophages.

The ganglioside composition of endotoxin-responsive C3H/HeN murine peritoneal macrophages is known to undergo dramatic changes in vivo in response to intraperitoneal lipopolysaccharides (LPS), unlike endotoxin-hyporesponsive C3H/HeJ macrophages. To better investigate the mechanism behind LPS-induced macrophage ganglioside changes, resident C3H/HeN peritoneal macrophages were treated in vitro with 0.1-1.0 micrograms/ml LPS for 6-96 hr, but showed no differences in membrane ganglioside patterns. Coincubation of macrophages with lymphocytes and treating with LPS again elicited no ganglioside changes. In contrast, interferon gamma (IFN-gamma)-primed macrophages showed a dramatic shift in intensity of one ganglioside when treated with LPS in vitro; an additional macrophage ganglioside appeared when IFN-gamma-primed, LPS-treated macrophages were coincubated with lymphocytes. Ganglioside expression induced in vitro still did not approach the complex changes seen in vivo. However, transplanting C3H/HeN macrophages intraperitoneally into C3H/HeJ mice, followed by administration of intraperitoneal LPS, did reveal striking changes in ganglioside expression that resembled the pattern seen in vivo. Thus, LPS alone does not provide the necessary direct signal to promote macrophage ganglioside change even though it alters macrophage function. IFN-gamma appears to be one important mediator; however, complex interactions involving other cytokines or migration of independent populations of mononuclear cells may be required for the full manifestation of LPS-induced ganglioside expression in macrophages.

Comparison of thymocyte and T lymphocyte gangliosides from C3H/HeN and C3H/HeJ mice.

Gangliosides have been prepared from resting murine thymocytes and splenic T cells. Profoundly different two-dimensional thin layer chromatography (2D TLC) patterns were observed between these two cell types. Thymocytes contained 28-30 discrete gangliosides of which eight represented major gangliosides. Splenic T lymphocytes from both strains had much simpler patterns, with six to seven major gangliosides and 12-13 minor gangliosides. Computerized analysis of the thymocyte ganglioside patterns between LPS-responder C3H/HeN mice and lipopolysaccharide (LPS)-hyporesponsive C3H/HeJ mice revealed no significant difference in the major gangliosides. However, with splenic T cell gangliosides, there is a striking difference in the relative proportion of three homologous gangliosides between the two strains. Consistent with previous observations on macrophage gangliosides, the ratio of N-acetylneuraminic acid-containing ganglioside to N-glycolylneuraminic acid-containing ganglioside was higher in both thymocytes and T-cells from the LPS-responder strain. These results show that sialic acid-containing glycolipids from thymocytes and T lymphocytes between endotoxin responder and hyporesponder strains manifest small but significant changes. These differences are present in unstimulated cell populations and may represent a manifestation of the Lps gene.

Haemophilus aphrophilus sternal osteomyelitis.

We have described a unique case of Haemophilus aphrophilus sternal osteomyelitis, with two separate infections seven years apart. Historical data suggest dental abscesses and trauma, with hematogenous seeding, as the cause. The patient responded well to surgical debridement and penicillin therapy after dental extractions.

Fever, petechiae, and pulmonary infiltrates in an immunocompromised Peruvian man.

The diagnostic considerations raised by immunocompromised patients with opportunistic infection continue to expand. When such patients harbor latent or persistent infection acquired in a tropical environment, the diagnostic challenge is even greater. The Infectious Disease Service at Yale-New Haven Hospital was asked to see a middle-aged man from Peru with known T-cell lymphoma who had recently completed a course of chemotherapy. He presented to the hospital with fever, petechial skin rash, pulmonary infiltrates, and neutropenia. Ultimately this case illustrated the necessity for careful evaluation of such patients, looking, in particular, for evidence of opportunistic parasitic infection.