[Update on protein analysis of fixed tissues]. / Neues zur Proteinanalytik archivierter Gewebeproben.

Tissue samples have been routinely used for decades to distinguish healthy from diseased tissue in histopathological characterization. While nucleic acid-based methodologies have been successfully in use for many years, protein-based techniques, in contrast, are at a very early stage (with the exception of immunohistochemistry). One reason for this delay may be that the scientific community has long thought that formalin-fixed and paraffin embedded (FFPE) tissues are unfit for protein analysis. However, recent reports demonstrate that many protein methods that are routinely used for frozen tissues can also be applied for FFPE tissues, including Western blot, protein microarray, matrix-assisted laser desorption/ionization (MALDI) imaging and 2D gel electrophoresis. The present article provides an overview of recent developments in this field, focussing particular attention on quantitative analysis and high throughput technologies that have the potential to be integrated into the routine workflow of clinical pathology laboratories.

The role of CD4+ T cells in the protective immune response to Plasmodium chabaudi in vivo.

CD4+ T cells are an essential component of the protective immune response to Plasmodium chabaudi. In order to determine whether the presence of CD4+ T cells is necessary throughout a primary infection for a protective immune response to develop mice were depleted of their CD4+ T cells in vivo by treatment with specific antibodies. Removal of CD4+ T cells during the acute phase of infection renders mice incapable of clearing their infection. In contrast, removal of CD4+ T cells after this time did not affect their ability to control their parasitaemia. The ability to control parasitaemia correlated with appearance of malaria-specific IgG antibodies. Our data, therefore, suggest a mechanism requiring the presence of CD4+ T cells during the acute pre-IgG period. Later, after IgG has been produced, this mechanism is no longer required.

Analysis of T lymphocyte reactivity to complex antigen mixtures by the use of proteins coupled to latex beads.

This report describes a suitable model for analysing heterogeneous T cell responses to complex foreign antigens using coated polystyrene beads. The advantage of this technique is that it allows the simple removal of detergents from bound antigen so that biochemically separated antigens or crude antigen mixtures can be used. Furthermore, due to the enhanced uptake of latex-bound antigens by phagocytic antigen-presenting cells (APC), very small amounts of antigen will suffice for activation of T cells in vitro. The potential use of this technique to analyse relevant T cell responses to antigens which are difficult to obtain purified in bulk quantities, is discussed.

Denitrification at a long-term forested land treatment system in the Piedmont of Georgia.

Spray irrigation of forested land can provide an effective system for nutrient removal and treatment of municipal wastewater. Evolution of N2 + N2O from denitrifying activity is an important renovation pathway for N applied to forested land treatment systems. Federal and state guidance documents for design of forested land treatment systems indicate the expected range for denitrification to be up to 25% of applied N, and most forest land treatment systems are designed using values from 15 to 20% of applied N. However, few measurements of denitrification following long-term wastewater applications at forested land treatment sites exist. In this study, soil N2 + N2O-N evolution was directly measured at four different landscape positions (hilltop, midslope, toe-slope, and riparian zone) in a forested land treatment facility in the Georgia Piedmont that has been operating for more than 13 yr. Denitrification rates within effluent-irrigated areas were significantly greater than rates in adjacent nonirrigated buffer zones. Rates of N2 + N2O-N evolved from soil in irrigated forests ranged from 5 to 10 kg ha(-1) yr(-1) N on the three upland landscape positions and averaged 38 kg ha(-1) yr(-1) N within the riparian zone. The relationship between measured riparian zone denitrification rates and soil physical and chemical properties was poor. The best relationship was with soil temperature, with an r2 of 0.18. Overall, on a landscape position weighted basis, only 2.4% of the wastewater-applied N was lost through denitrification.

Super-resolution segmentation of imaging mass spectrometry data: Solving the issue of low lateral resolution.

In the last decade, imaging mass spectrometry has seen incredible technological advances in its applications to biological samples. One computational method of data mining in this field is the spatial segmentation of a sample, which produces a segmentation map highlighting chemically similar regions. An important issue for any imaging mass spectrometry technology is its relatively low spatial or lateral resolution (i.e. a large size of pixel) as compared with microscopy. Thus, the spatial resolution of a segmentation map is also relatively low, that complicates its visual examination and interpretation when compared with microscopy data, as well as reduces the accuracy of any automated comparison. We address this issue by proposing an approach to improve the spatial resolution of a segmentation map. Given a segmentation map, our method magnifies it up to some factor, producing a super-resolution segmentation map. The super-resolution map can be overlaid and compared with a high-res microscopy image. The proposed method is based on recent advances in image processing and smoothes the "pixilated" region boundaries while preserving fine details. Moreover, it neither eliminates nor splits any region. We evaluated the proposed super-resolution segmentation approach on three MALDI-imaging datasets of human tissue sections and demonstrated the superiority of the super-segmentation maps over standard segmentation maps.

CD4+ T cells and B cells are necessary for the transfer of protective immunity to Plasmodium chabaudi chabaudi.

It is shown here that B cells, in addition to CD4+ T cells, are necessary for the development of protective immunity to Plasmodium chabaudi chabaudi (P. chabaudi) in mice. Reconstitution of severe combined immunodeficient (SCID) mice with immune or normal CD4+ T cells protected the majority of mice against an otherwise lethal challenge but the mice were unable to clear their parasitemias. By contrast, transfer of the same T cell populations into athymic nu/nu mice enabled the recipients to control and clear their infections, immune CD4+ T cells being most effective. Furthermore, SCID mice given CD4+ T cells from immune and normal donors simultaneously with immune B cells also could eliminate their infection. Clearance of parasitemia correlated with the presence of malaria-specific antibodies in the serum. The role of B cells and CD4+ T cells in the protective immune response to P. chabaudi is discussed.

CD4+T cell-dependent effector mechanisms important in the immune response to the erythrocytic stages of Plasmodium chabaudi chabaudi (AS).

The ability to produce IFN-gamma during an erythrocytic infection with P. chabaudi chabaudi is not an indicator of resistance or susceptibility of mice to this infection. However, IFN-gamma may play a transient role in immune control of the parasite. We have shown that after its removal in vivo by antibody treatment the acute phase of infection is clearly exacerbated. After the acute phase of infection the T cell response changes from one characterized by IFN-gamma production to one of a T helper response for antibody production. The IFN-gamma response to parasite challenge can be recovered if the challenge infection is given many weeks after the mice have cleared their primary parasitaemias. These data suggest that the CD4+ T cell response is a heterogenous one and regulated by its cytokine production. The protective mechanisms therefore probably reflect the nature of the ongoing immune response.

Gamma interferon production in endotoxin-responder and -nonresponder mice during infection.

The production of gamma interferon (IFN-gamma) in response to infection and to a number of other agents was compared in Lpsn (C3H/HeN and C57BL/10ScSn) and Lpsd (C3H/HeJ and C57BL/10ScCr) mouse strains. Large differences in IFN-gamma production were observed between C57BL/10ScCr mice and the other mouse strains. With the exception of C57BL/10ScCr, all mouse strains, including C3H/HeJ, exhibited transient levels of IFN-gamma during infection with Salmonella typhimurium. Spleen cells of these mice, explanted on day 3 of infection, produced in vitro IFN-gamma spontaneously; this production was enhanced considerably by heat-killed S. typhimurium, heat-killed Propionibacterium acnes, concanavalin A (ConA), or lipopolysaccharide (LPS). These stimuli, except for LPS, also induced IFN-gamma production in cultures of normal spleen cells from noninfected animals. In contrast, C57BL/10ScCr mice produced no IFN-gamma following infection with S. typhimurium. Also, spleen cells of these mice, explanted on day 3 of infection, exhibited no spontaneous IFN-gamma production. A marginal response was obtained by additional stimulation of the cells with killed S. typhimurium, and a moderate response was obtained with ConA. Normal spleen cells from noninfected C57BL/10ScCr mice showed no IFN-gamma response to killed S. typhimurium, killed P. acnes, or LPS and only a low response to ConA. Impaired IFN-gamma production in C57BL/10ScCr mice was also evident during infection with Plasmodium chabaudi chabaudi, with which a low IFN-gamma response was seen only occasionally. Also, spleen cells from infected animals (days 2 to 8 after infection) exhibited only a very low level of IFN-gamma production in vitro; however, this production could be enhanced further by ConA. In comparison, C57BL/10ScSn mice infected with P. chabaudi chabaudi produced significant amounts of IFN-gamma. Spleen cells explanted from infected animals produced IFN-gamma spontaneously in vitro; this production was enhanced further by killed P. acnes and ConA. The results showed that in addition to the defect in LPS responsiveness, C57BL/10ScCr mice possess a defect in IFN-gamma production in response to different stimuli. During infection, IFN-gamma production and sensitization to LPS occurred in parallel. Infected Lpsn mice exhibited enhanced sensitivity and infected Lpsd C3H/HeJ mice exhibited reasonable sensitivity to the lethal effects of LPS. Lpsd C57BL/10ScCr mice remained resistant to LPS when infected with S. typhimurium and exhibited only marginal sensitivity when infected with P. chabaudi chabaudi.

The response of CD4+ T cells to Plasmodium chabaudi chabaudi.

We have studied the role of CD4+ T cells in the immune response to Plasmodium chabaudi chabaudi. From in vivo experiments in which the different subsets of T cells were depleted, it is clear that CD4+ T cells are essential for the generation of protective immunity. Our limiting dilution analysis show that the CD4 T-cell response to P. chabaudi antigens is heterogeneous, in that distinct functions can be performed by different responding T cells, and these responses change during infection. During the first phase of the infection the predominant response is that of a TH1-type cell, producing IL-2 and IFN-gamma. This correlates with the appearance of IFN-gamma in the serum of infected animals. After the clearance of the acute parasitemia, i.e. in the second phase of the infection, the specific response is characterised by TH2 cells, which are effective helper cells for antibody production and presumably are necessary for the switch of IgM to IgG. CD4+ T cells are effector cells are not necessary in the second phase of the infection; mice which have been depleted of CD4+ T cells at this time are able to control their infection in a manner similar to untreated mice. This ability to control parasitemia coincides with the production of specific IgG but not IgM antibodies and the predominance of TH2 type helper cells. Therefore, our data suggest that malaria-specific IgG antibodies are important effectors in the second phase of an infection with P. chabaudi chabaudi.

Role of gamma interferon during infection with Plasmodium chabaudi chabaudi.

A role has been proposed for inflammatory mediators such as gamma interferon (IFN-gamma) and reactive oxygen intermediates in the control of the blood stages of Plasmodium organisms. It was previously shown that IFN-gamma can be detected in the plasma of mice with a primary infection by Plasmodium chabaudi chabaudi (AS). We found that susceptible and other resistant mouse strains produced IFN-gamma, suggesting that susceptibility is not due to a defect in IFN-gamma production. Administration of IFN-gamma to intact C57BL/6 mice slightly decreased and partially delayed parasitemia, whereas in vivo depletion of IFN-gamma through injection of a "cocktail" of monoclonal antibodies against IFN-gamma exacerbated infection. Since CD4+ T cells are essential for the development of a protective immune response to P. chabaudi chabaudi, we tested whether CD4+ T cells are responsible for IFN-gamma production in vivo and whether exogenous IFN-gamma can replace the protective function of the CD4+ T cells. Mice depleted of CD4+ T cells were unable to produce IFN-gamma, but factors in addition to IFN-gamma may be important in parasite clearance.