Elimination of the non-specific binding of avidin to tissue sections

Summary A simple procedure is described for eliminating non-specific staining with avidin—peroxidase conjugates. Murine ovaries were embedded in either paraffin wax or epoxy resin and, after blocking endogenous peroxidase activity, were treated with 10 µg/ml biotinylatedPisum sativum agglutinin. Avidin—peroxidase conjugates (5 µg/ml), diluted in standard 0.05m tris-buffered saline, pH 7.6, containing 0.139m NaCl, produced considerable background coloration and intense mast cell staining in controls without the lectin. This background diminished as the ionic strength of the buffer was raised. At 0.125m Tris-buffered saline (containing 0.347m NaCl) the background was completely unstained, with elimination of all binding to mast cells and only minimal loss of specific lectin binding.     

An anti-inflammatory role for V alpha 14 NK T cells in Mycobacterium bovis bacillus Calmette-Guérin-infected mice

The possible contribution of NKT cells to resistance to Mycobacterium tuberculosis infection remains unclear. In this paper we characterized the Valpha14 NKT cell population following infection with Mycobacterium bovis bacillus Calmette-Guérin (BCG). BCG infection determined an early expansion of Valpha14 NKT cells in liver, lungs, and spleen, which peaked on day 8 and was sustained until day 30. However, an NK1.1(+) Valpha14 NKT population preferentially producing IFN-gamma predominated at an early stage (day 8), which was substituted by an NK1.1(-) population preferentially producing IL-4 at later stages (day 30). Despite the fact that Valpha14 NKT cell-deficient mice eliminated BCG as did control mice, they had significantly higher numbers of granulomas in liver and lungs. Additionally, while control mice developed organized small granulomas, those in Valpha14 NKT-deficient mice had signs of caseation, large cellular infiltrates, and some multinucleated macrophages, suggesting that Valpha14 NKT cells may actually work as anti-inflammatory cells by limiting excessive lymphocyte influx and tissue pathology. In agreement, we found an increased spontaneous production and mRNA expression of TNF-alpha in liver and lungs of Valpha14 NKT-deficient mice, whose neutralization in vivo by anti-TNF-alpha mAbs consistently reduced the number of granulomas in liver and lungs. Together, our results support a regulatory role for Valpha14 NKT cells in the course of BCG infection through their ability to limit the extent of inflammatory response and point to an important role for this cell subset as a regulator of the balance between protective responses and immunopathology.     

Cholera toxin B pretreatment of macrophages and monocytes diminishes their proinflammatory responsiveness to lipopolysaccharide

The cholera toxin B chain (CTB) has been reported to suppress T cell-dependent autoimmune diseases and to potentiate tolerance of the adaptive immune system. We have analyzed the effects of CTB on macrophages in vitro and have found that preincubation with CTB (10 microg/ml) suppresses the proinflammatory reaction to LPS challenge, as demonstrated by suppressed production of TNF-alpha, IL-6, IL-12(p70), and NO (p < 0.01) in cells of macrophage lines. Pre-exposure to CTB also suppresses LPS-induced TNF-alpha and IL-12(p70) formation in human PBMC. Both native and recombinant CTB exhibited suppressive activity, which was shared by intact cholera toxin. In cells of the human monocyte line Mono Mac 6, exposure to CTB failed to suppress the production of IL-10 in response to LPS. Control experiments excluded a role of possible contamination of CTB by endotoxin or intact cholera toxin. The suppression of TNF-alpha production occurred at the level of mRNA formation. Tolerance induction by CTB was dose and time dependent. The suppression of TNF-alpha and IL-6 production could be counteracted by the addition of Abs to IL-10 and TGF-beta. IFN-gamma also antagonized the actions of CTB on macrophages. In contrast to desensitization by low doses of LPS, tolerance induction by CTB occurred silently, i.e., in the absence of a measurable proinflammatory response. These findings identify immune-deviating properties of CTB at the level of innate immune cells and may be relevant to the use of CTB in modulating immune-mediated diseases.     

Early stages of the apoptotic pathway in plant cells are reversible

Chromatin condensation and nDNA fragmentation, indicators of apoptosis in mammalian cells, occur in plant cells during senescence and following induction by chemical agents. In Nicotiana plumbaginifolia cells, camptothecin, okadaic acid, salicylic acid, hydrogen peroxide, and the calcium ionophore A23187 induced chromatin condensation and nDNA fragmentation. Exposure of cells to low concentrations or removal of the chemical agent resulted in an initial phase of chromatin condensation, followed by its reversal. A further feature of apoptosis in mammalian cells, annexin V binding, indicative of phosphotidylserine exposure, was also confirmed in relation to the other events in the apoptotic pathway. With respect to flow cytometric characteristics, apoptosis triggered by a variety of chemicals occurs in plant cells in a manner closely related to that in mammalian cells. However, the extent of chromatin condensation is substantially greater, and in the early stages is reversible.     

Gender-Specific Association between Tobacco Smoking and Central Obesity among 0.5 Million Chinese People: The China Kadoorie Biobank Study

Objectives

Lifestyle factors are well-known important modifiable risk factors for obesity; the association between tobacco smoking and central obesity, however, is largely unknown in the Chinese population. This study examined the relationship between smoking and central obesity in 0.5 million Chinese adults, a population with a low prevalence of general obesity, but a high prevalence of central obesity.

Subjects

A total of 487,527 adults (200,564 males and 286,963 females), aged 30-79 years, were enrolled in the baseline survey of the China Kadoorie Biobank (CKB) Study conducted during 2004-2008. Waist circumference (WC) and WC/height ratio (WHtR) were used as measures of central obesity.

Results

The prevalence of regular smokers was significantly higher among males (60.6%) than among females (2.2%). The prevalence of central obesity increased with age and BMI levels, with a significant gender difference (females>males). Of note, almost all obese adults (99.4%) were centrally obese regardless of gender. In multivariable regression analyses, adjusting for age, education, physical activity, alcohol use and survey site, regular smoking was inversely associated with BMI in males (standardized regression coefficients, β= -0.093, p<0.001) and females (β= -0.025, p<0.001). Of interest, in the BMI stratification analyses in 18 groups, all βs of regular smoking for WHtR were positive in both genders; the βs showed a significantly greater increasing trend with increasing BMI in males than in females. In the analyses with model adjustment for BMI, the positive associations between regular smoking and WHtR were stronger in males (β= 0.021, p<0.001) than in females (β= 0.008, p<0.001) (p<0.001 for gender difference). WC showed considerably consistent results.

Conclusions

The data indicate that tobacco smoking is an important risk factor for central obesity, but the association is gender-specific and depends on the adjustment for general obesity.     

Efficient transient transformation of suspension culture-derived apple protoplasts

A protocol for comparatively efficient delivery of plasmid DNA into suspension culture-derived apple protoplasts is described. Increases in efficiency of transfection are achieved by elevating the temperature during protoplast exposure to the DNA. Two methods to detect transformed protoplasts are compared and the effectiveness and ease of application of flow cytometry techniques to this high throughput method is discussed.