Macroscopic biofilms in fracture-dominated sediment that anaerobically oxidize methane.

Methane release from seafloor sediments is moderated, in part, by the anaerobic oxidation of methane (AOM) performed by consortia of archaea and bacteria. These consortia occur as isolated cells and aggregates within the sulfate-methane transition (SMT) of diffusion and seep-dominant environments. Here we report on a new SMT setting where the AOM consortium occurs as macroscopic pink to orange biofilms within subseafloor fractures. Biofilm samples recovered from the Indian and northeast Pacific Oceans had a cellular abundance of 10(7) to 10(8) cells cm(-3). This cell density is 2 to 3 orders of magnitude greater than that in the surrounding sediments. Sequencing of bacterial 16S rRNA genes indicated that the bacterial component is dominated by Deltaproteobacteria, candidate division WS3, and Chloroflexi, representing 46%, 15%, and 10% of clones, respectively. In addition, major archaeal taxa found in the biofilm were related to the ANME-1 clade, Thermoplasmatales, and Desulfurococcales, representing 73%, 11%, and 10% of archaeal clones, respectively. The sequences of all major taxa were similar to sequences previously reported from cold seep environments. PhyloChip microarray analysis detected all bacterial phyla identified by the clone library plus an additional 44 phyla. However, sequencing detected more archaea than the PhyloChip within the phyla of Methanosarcinales and Desulfurococcales. The stable carbon isotope composition of the biofilm from the SMT (-35 to -43‰) suggests that the production of the biofilm is associated with AOM. These biofilms are a novel, but apparently widespread, aggregation of cells represented by the ANME-1 clade that occur in methane-rich marine sediments.

Cytomegalovirus induces sialyl Lewis(x) and Lewis(x) on human endothelial cells.

BACKGROUND: Cytomegalovirus (CMV) is the primary viral cause of complications in transplant recipients. We sought to understand the mechanisms of its dissemination and induction of vascular disease, which may lead to transplant complications. Sialyl Lewis(x) (sLe(x)) and Lewis(x) (Le(x)) are known for their roles in mediating cell adhesion and as tumor-associated carbohydrate antigens. Herein we explore whether CMV induces surface expression of these important molecules in endothelial cells (EC). METHODS: Flow cytometry was used to detect surface expression of sLe(x) and Le(x) on CMV-infected human umbilical vein endothelial cells (HUVEC), with or without ultraviolet inactivation of the virus. To elucidate mechanisms of CMV-mediated induction, mRNA coding for predominant HUVEC sialyltransferases (ST) and fucosyltransferases (FT), key enzymes in sLe(x) and Le(x) synthesis, was analyzed by Northern blot. Dual immunohistochemical staining for sLe(x) and Le(x) expression of human colon and placental tissue was performed to investigate in vivo relevance. RESULTS: sLe(x) expression on CMV-infected HUVEC was strongly up-regulated by 8 days after inoculation. Le(x) expression was detectable earlier and increased steadily over time. In contrast, ultraviolet-inactivated CMV did not induce expression of these molecules. Northern blot assays demonstrated higher levels of important EC glycosyltransferases ST-IV, FT-III, and FT-IV in CMV-infected EC. Finally, high levels of sLe(x) and Le(x) were expressed in CMV-infected EC in vivo. CONCLUSIONS: Given the known biologic functions of sLe(x) and Le(x), we suggest that CMV induction of these molecules may have widespread consequences ranging from CMV dissemination to induction of CMV-associated vascular disease, including thrombosis.

Attenuation of cytomegalovirus-induced endothelial intercellular adhesion molecule-1 mRNA/protein expression and T lymphocyte adhesion by a 2'-O-methoxyethyl antisense oligonucleotide.

BACKGROUND: Intercellular adhesion molecule-1 (ICAM-1) is strongly induced under inflammatory conditions associated with allograft rejection, thereby promoting leukocyte recruitment and activation at the site of inflammation. Enhancement of ICAM-1 expression can also be the result of viral infection, in particular human cytomegalovirus (CMV), a frequent source of complications in the transplant recipient. In vitro studies have shown that CMV infection of endothelial cells (EC) results in the direct enhancement of ICAM-1 expression and consequent leukocyte adhesion/activation suggesting mechanisms by which CMV exacerbates graft vascular disease. Although treatment of EC with ICAM-1-specific antisense oligonucleotides has been shown to attenuate ICAM-1 induction under simulated inflammatory conditions (i.e., TNF-alpha), no studies have addressed their effectiveness on virally-induced ICAM-1 expression. RESULTS: In the current investigation, we show that the progressive increase in endothelial ICAM-1 protein expression that follows inoculation with CMV correlates with a progressive accumulation of ICAM-1 mRNA. Furthermore, we demonstrate that treatment of EC with a partially 2'-O-methoxyethyl modified ICAM-1-specific antisense oligonucleotide before viral inoculation significantly reduces CMV-associated induction of ICAM-1 protein and mRNA expression. Finally, we show that antisense-mediated attenuation in ICAM-1 expression results in a significant reduction of T lymphocyte adhesion to CMV-infected EC monolayers, an interaction that has been implicated in allogeneic T lymphocyte activation, in viral transmission to transiently adherent leukocytes and subsequent hematogenous dissemination. CONCLUSIONS: These findings demonstrate for the first time that antisense oligonucleotides can effectively reverse virally-induced host cellular protein expression, specifically ICAM-1, as well as consequent T lymphocytes adhesion, thus broadening the potential clinical utility of antisense oligonucleotides.

Endotoxin in middle-ear effusions from patients with chronic otitis media with effusion.

Endotoxin concentrations were determined in middle-ear effusions (MEEs) from 89 children with chronic otitis media by using the Limulu's amoebocyte lysate assay. Mean concentrations of endotoxin in Haemophilus influenzae-positive and Streptococcus pneumoniae-positive MEEs were 157 and 21.8 ng/ml, respectively, and were significantly different (P less than 0.01). Endotoxin was also found in Gram stain-positive, culture-negative and Gram stain-negative, culture-negative MEEs, but the levels were not significantly different (P greater than 0.05). However, the endotoxin concentrations in both groups of culture-negative MEEs significantly lower than those found in MEEs that grew either H. influenzae or S. pneumoniae (P less than 0.05). These results show that endotoxin is present in a high percentage of human MEEs, including those that are culture negative, and may contribute to the pathogenesis of otitis media with effusion.

Experimental otitis media with effusion induced by nonviable Hemophilus influenzae: cytologic and histologic study.

In an earlier study the authors demonstrated that formalin-killed Hemophilus influenzae induces serous-type middle ear effusion in chinchillas and provides an excellent model for the study of human otitis media with effusion. The present study was initiated to evaluate the morphologic and histologic changes that occur in the middle ear after injection of this organism. All of the experimental animals injected with formalin-killed H. influenzae in the present study had straw-colored serous-type effusions within four days after injection. The submucosal thickness, mononuclear cell density, and capillary permeability all increased dramatically in the experimental animals. Marked bleeding, tissue edema, and cellular infiltration in the submucosa were prominent findings after injection of the inactivated bacteria. Half of the experimental animals had histologic evidence of marked proliferation of epithelial cells resembling adhesive otitis media. These findings suggest that nonviable H. influenzae are capable of inducing severe inflammatory changes in the middle ear and may play an important role in the pathogenesis of otitis media with effusion and its sequelae.

Experimental otitis media with effusion following middle ear inoculation of nonviable H influenzae.

In order to test the hypothesis that nonviable bacteria can induce middle ear inflammation leading to persistent middle ear effusion (MEE), we conducted an animal experiment using formalin-killed Hemophilus influenzae, the bacterium reported to be the most common pathogen isolated from chronic MEEs. Over 70% of the chinchillas injected with formalin-killed H influenzae type b or a nontypeable isolate developed sterile, straw-colored serous MEEs, and exhibited histological evidence of extensive inflammatory changes of the middle ear mucosal connective tissue and epithelium. Control animals injected with pyrogen-free sterile saline did not exhibit any inflammatory changes or effusions in the middle ears. Our data suggest that endotoxin on the surface of H influenzae, a gram-negative bacterium, may be responsible for the induction of the otitis media with effusion. It is suggested that endotoxin (even when the organisms are no longer viable) may be responsible for the production of serous MEE and inflammatory changes in the middle ear.

Experimental otitis media in chinchillas. I. Baseline immunological investigation.

Baseline immunological data on the chinchilla were obtained, and a detailed method for purification of each immunoglobulin is described. Antiserum to each chinchilla immunoglobulin was made by immunizing rabbits with immunoglobulins purified from serum and colostrum. The main classes of serum immunoglobulins in the chinchilla are G, A, and M, similar to those in other rodents and humans. The immunohistochemical study showed that the distribution of immunocytes in lymphoid organs is similar to that of other rodents and humans. In the eustachian tube the glandular acinar cells were distinctly positive for IgA staining. A few IgA-forming cells and very rare IgG- and IgM-forming cells were found in the eustachian tube mucosa, but all were very rare in the middle ear mucosa. These findings suggest that the secretory immune system of the chinchilla eustachian tube may be active in the normal state and that the local immune system of the normal middle ear mucosa may be latent but could become active following antigenic stimulation.

The histocompatibility (HLA) antigen distribution in diabetes in southern African Blacks (Xhosa).

The frequency distributions of HLA antigens in 25 juvenile-onset diabetics (JOD) and 56 maturity-onset diabetics (MOD) belonging to a southern African black tribe (Xhosa) were compared with those of 153 non-diabetic Xhosa blacks. Unlike the findings in white JODs, there was no increase of B8 or B15 nor a reduced frequency of B7 but an apparently, significantly increased frequency of Bw35 and A2 in both Xhosa JODs and Xhosa MODs respectively. This is the first ethnic group in which an HLA antigen marker has been found for MOD. Furthermore, these findings suggest that diabetes, both JOD and MOD, in white people is a different genetic disease from the diabetes among the Xhosa tribe.

Effect of cefamandole nafate on blood coagulation and platelet function.

Platelet function and coagulation were tested after brief in vivo exposure to cefamandole nafate and in the presence of various concentrations of the drug in vitro. Alterations in hemostatic function were observed only with exposure to drug concentrations severalfold higher than those expected to prevail clinically.