Analysis of the fine specificity and cross-reactivity of monoclonal anti-lipid A antibodies

We have investigated the fine specificity of anti-lipid A antibodies to identify conserved lipid A antigens. Because lipid A derived from many different Gram-negative bacteria has similar biologic activities, the conserved regions may be of particular importance for the immunostimulatory and toxic properties of lipid A. We found that five of nine antibodies bound to a wide variety of Gram-negative bacteria. All these widely cross-reactive antibodies bound to the same antigenic site within lipid A. Polymyxin B, an inhibitor of lipid A activity, bound to this site as well. The widely cross-reactive antibodies bound to native and base-hydrolyzed lipid A equally well, and also bound to the monosaccharide precursor lipid X. The less cross-reactive antibodies recognized base-hydrolyzed lipid A poorly, and did not recognize lipid X at all. Other investigators have shown that lipid X has some of the activities of lipid A in vitro and can inhibit the lethal toxicity of LPS in vivo. On the basis of this study, we suggest that lipid X contains a conserved lipid A epitope as well.     

Assay of reducing sugars in the nanomole range with 2,2'-bicinchoninate

The photometric assay of reducing sugars with 2,2'-bicinchoninate was improved and its conditions were optimized. Optical density is linear between 1 and 25 nmol sugar/sample, and the assay is not affected by borate, phosphate, or other buffer anions. The molar extinction coefficients produced by the standard procedure with the 12 most common monosaccharides occurring in carbohydrates and conjugated glycocompounds are listed.     

Antigen presentation by the BCL1 murine B cell line: in vitro stimulation by LPS

We examined the antigen-presenting capacity of BCL1 tumor cells, which are capable of differentiating in vitro with respect to immunoglobulin synthesis/secretion under the influence of LPS. In vivo passaged BCL1 cells depleted of host cell contamination either by positive selection employing panning with anti-lambda reagents, or by elimination of latex-ingesting adherent cells, are capable of MHC-restricted antigen presentation to a GAT-immune T cell line. The BCL1 cells act as antigen-presenting cells when freshly explanted, but gradual loss of this function occurs, and cells cultured for 3.5 days cannot present antigen unless LPS is included during the culture period. BCL1 cells are equivalently Ia+ after the culture period with or without LPS stimulation. Other B cell lines capable of antigen presentation appear to express this trait constitutively, and the in vivo passaged BCL1 line is therefore unique among B cell lines in having antigen-presenting cell function that can be modulated. The data suggest that freshly explanted or LPS-cultured BCL1 cells are heterogeneous with respect to antigen-presenting capacity, and the basis for this heterogeneity is being sought. BCL1 offers an opportunity to study requirements for antigen presentation by B cells.     

Treatment of methanol poisoning with ethanol and hemodialysis.

Twelve cases of methanol poisoning are reviewed. The clinical presentation and biochemical features are described and the results of treatment with alkali, ethanol and dialysis reported. The outcome of methanol poisoning appears to be related more to the interval between the time of ingestion and the start of therapy and to the degree of acidosis than to the initial serum methanol level. Therefore, early and aggressive treatment with bicarbonate and ethanol and subsequent institution of hemodialysis are strongly recommended whenever methanol can be detected in the blood, especially when metabolic acidosis of the anion-gap type is present, when mental or visual disturbances are present, or when more than 30 ml of absolute methanol has been consumed.     

Population structure of the intertidal copepod Tigriopus californicus as revealed by field manipulation of allele frequencies

Summary Allele frequencies in natural T. californicus populations were perturbed by introduction of copepods from neighboring differentiated populations. In five experiments, the Gpt ^F allele was introduced into single recipient pools at a frequency of approximately 20%. In each case, the introduced allele declined to low frequencies (<3%) in less than one month, apparently due to dilution by residents of other pools on the same outcrop. In a larger scale experiment, the Pgi ^F was introduced into four pools on a single small rock outcrop; all pools on the outcrop were subsequently monitored. While the allele frequency fell from approximately 40% to 10% during the first six weeks after the transplant, no further change in frequency was observed for the duration of the experiment (16 months). Within six weeks some spread of the allele to non-recipient pools on the same outcrop was observed; by eight months, allele frequencies in all pools on the outcrop were similar. Hence, despite the extensive turnover of subpopulations as single pools evaporate or are washed out, genetic homogeneity and stability of entire outcrops are maintained via extensive inter-pool gene flow; this contrasts sharply with the highly restricted levels of inter-outcrop gene flow.     

Blocking of acidosis-mediated apoptosis by a reduction of lactate dehydrogenase activity through antisense mRNA expression.

Lactic acid produced from the cells is a potential cause of extra- and intracellular acidification. Due to scarce technical tools, lactic acid that leads to acidification could not be reduced and direct evidence of the relationship between metabolic lactate and apoptosis has not yet been elucidated. In this study, we designed a cellular pH regulation system in CHO cells by a reduction of lactate dehydrogenase (LDH) activity through LDH antisense mRNA expression. This inhibited lactate production and, therefore, acidification of the cytosol. Under HCO3(-)-buffered growth conditions, both the parent CHO cells and the engineered CHO cells maintained their extracellular pH and intracellular pH fairly well. However, upon acidification of the cytosol, only the parent CHO cells underwent apoptosis under HCO3(-)-free conditions. In fact, we observed a number of apoptosis-related events only in control cells, including mitochondrial dysfunction, cytochrome c release, and an increase in caspase-3 enzymatic activity.