Expression of eight different chitinase genes, representing members of five chitinase classes, was studied in Medicago truncatula roots during formation of arbuscular mycorrhiza with Glomus intraradices, nodulation with Rhizobium meliloti, and pathogen attack by Phytophthora megasperma f. sp. medicaginis, Fusarium solani f. sp. phaseoli (compatible interactions with root rot symptoms), Ascochyta pisi (compatible, symptomless), and F. solani f. sp. pisi (incompatible, nonhost interaction). In the compatible plant-pathogen interactions, expression of class I, II, and IV chitinase genes was enhanced. The same genes were induced during nodulation. Transcripts of class I and II chitinase genes accumulated transiently during early stages of the interaction, and transcripts of the class IV chitinase gene accumulated in mature nodules. The pattern of chitinase gene expression in mycorrhizal roots was markedly different: Expression of class I, II, and IV chitinase genes was not enhanced, whereas expression of three class III chitinase genes, with almost no basal expression, was strongly induced. Two of these three (Mtchitinase III-2 and Mtchitinase III-3) were not induced at all in interactions with pathogens and rhizobia. Thus, the expression of two mycorrhiza-specific class III chitinase genes can be considered a hallmark for the establishment of arbuscular mycorrhiza in Medicago truncatula.

A class III chitinase gene, Mtchitinase III‐3, has been found to be induced in roots of Medicago truncatula specifically upon colonization by the arbuscular mycorrhizal fungi Glomus mosseae and Glomus intraradices. Here we show by in situ hybridization techniques that Mtchitinase III‐3 is only expressed in cortical root cells containing arbuscules formed by either of the fungi. Transcripts of Mtchitinase III‐3 were detected in cells containing developing as well as mature arbuscules. They were absent from cells adjacent to arbuscule‐containing cells, from cells in contact with intraradical hyphae and from cortical cells in non‐colonized areas of the root. These results indicate that the product of the Mtchitinase III‐3 gene has a role within individual cortical cells during formation and functioning of arbuscules.

Lymphoid tissues cultured either as tissue blocks or as cell suspensions are used to study the behaviour of immune cells within their habitat. The preservation of tissue structures in tissue blocks, which is considered to be a major advantage, has been poorly defined. We characterised the morphological evolution of tissue cultures from human palatine tonsils and compared their lymphocyte subsets and the constitutive cytokine gene expression to those in autologous tonsillar single-cell suspension cultures over time, and after adding cyclosporin A (CsA) to mimic the situation in individuals treated with immunosuppressive drugs. Density and morphology of follicles were conserved up to 4 days, during which tissue cultures exhibited similar cell viability as suspension cultures, but a significantly less frequent increase of CD95 expression in T cells, smaller variation of the proportion of CD4 + cells and better CD21 + /CD23 À B-cell survival. Treatment with cyclosporin A at higher concentrations resulted in superior histologic preservation of lymphoid tissue structures and seemed to further prevent the expression of CD95 by CD3 + cells and the activation in tissue culture of CD21 + cells. Constitutive gene expression levels of the stromal cytokines interleukin (IL)-1h and interleukin-6 in tissue culture were significantly higher than those in suspension cultures. These results suggest that tonsillar tissue cultures preserve their structure only for a limited time, during which they more closely reflect processes in vivo, including a state of iatrogenic immunosuppression, than do their cell suspension counterparts. D

Summary• The association of plants with arbuscular mycorrhizal (AM) fungi is widespread in nature, but little is known about molecular aspects of this symbiosis. Particularly during the early stages of the AM symbiosis, it is difficult to monitor growth of the two partners, to dissect gene expression patterns and to correlate them with plant, fungal or symbiosis development.• A new system, the 'mini-mycorrhizotron', was established to cultivate seedlings of Medicago truncatula in mycorrhizal symbiosis with Glomus intraradices under gnotobiotic conditions. This system allows natural growth of the symbiotic partners and permits the continuous noninvasive observation of the development of plant and fungus under a microscope.• The mini-mycorrhizotron was used to determine the stage of induction of a mycorrhiza-related gene detected by differential display-reverse transcription-PCR, namely a novel chalcone synthase ( Mt-chs1 ). The gene is induced in roots at the stage of the first fungal contact.• The mini-mycorrhizotron allowed identification and cloning of a symbiosis-related gene, and the correlation between its expression and the developmental stage of the symbiosis was established. This provides a useful tool for molecular and developmental studies of the early stages of AM symbioses.

The cytokine response to invading microorganisms is critical for priming the adaptive immune response. During acute HIV infection, the response is disrupted, but the mechanism is poorly understood. We examined the cytokine response in human lymphoid tissue, acutely infected ex vivo with HIV. Lymphoid tissue was cultured either as blocks or as human lymphocyte aggregate cultures (HLAC) of tonsils and lymph nodes. This approach allowed us to examine the effects of HIV on cytokines using distinct culture techniques. In contrast to HLAC, mock-infected tissue blocks displayed a 50- to 100-fold up-regulation of mRNAs for IL-1β, -6, and -8 in the first 6 days of culture. Parallel increases were also noted at the protein level in the supernatants. Although IL-1β, -6, and -8 are known to synergistically enhance HIV replication, peak HIV replication (measured as p24 Ag) was similar in tissue blocks and HLAC. Surprisingly, vigorous HIV replication of CXCR4- and CCR5-tropic HIV strains did not result in characteristic mRNA profiles for IL-1β, -2, -4, -6, -8, -10, -12, -15, IFN-γ, TNF-α, TGF-β, and β-chemokines in tissue blocks or HLAC. The increased expression of IL-1β, -6, and -8 in tissue blocks may approximate clinical situations with heightened immune activation; neutralization of these cytokines resulted in inhibition of HIV replication, suggesting that these cytokines may contribute to HIV replication in certain clinical settings. These results also indicate that different molecular mechanisms govern HIV replication in tissue blocks and HLAC. Prevention of effective cytokine responses may be an important mechanism that HIV uses during acute infection.