Immunohistological localisation of two hydatid antigens (antigen 5 and antigen b) in the cyst wall, brood capsules and protoscoleces of Echinococcus granulosus (ovine and equine) and E. multilocularis using immunoperoxidase methods.

Cyst wall, brood capsules and evaginated protoscoleces of E. granulosus (ovine and equine) and E. multilocularis were fixed in 10% formol-saline, embedded in paraffin and cut at 8 micrometer. Specific rabbit antisera to antigen 5 and antigen B of hydatid cyst fluid were used with immunoperoxidase methods to localise the antigens in the histological sections. Antigen 5 was found in all parasites and was associated with cells of the subtegumental area of the protoscolex, the brood capsule wall and the germinal membrane. The labelled antigen appeared as distinct granules in all areas. It is suggested that antigen 5 may be synthesised in all of these sites and that a source of the antigen in cyst fluid may be the germinal and brood capsule membranes. The laminated membranes of E. granulosus (ovine and equine) were, except for the superficial layers, free from antigen 5. Antigen B was present in all parasites. It was distributed diffusely throughout the laminated membrane, germinal membrane and brood capsule wall. There were areas of densely labelled antigen B on the surface of the distal cytoplasm of the protoscolex tegument and the surface of calcareous corpuscles. The distribution of antigen B in relation to PAS positive material and possible complement activating substances is discussed. The laminated membrane of E. granulosus was apparently more permeable to antigen B than to antigen 5. It is suggested that differences in the diffusion of these antigens through the laminated membranes of hydatid cysts in the same or different host species may account for variable serological responses during infection.     

The granzyme B-Serpinb9 axis controls the fate of lymphocytes after lysosomal stress

Cytotoxic lymphocytes (CLs) contain lysosome-related organelles (LROs) that perform the normal degradative functions of the lysosome, in addition to storage and release of powerful cytotoxins employed to kill virally infected or abnormal cells. Among these cytotoxins is granzyme B (GrB), a protease that has also been implicated in activation (restimulation)-induced cell death of natural killer (NK) and T cells, but the underlying mechanism and its regulation are unclear. Here we show that restimulation of previously activated human or mouse lymphocytes induces lysosomal membrane permeabilisation (LMP), followed by GrB release from LROs into the CL cytosol. The model lysosomal stressors sphingosine and Leu-Leu-methyl-ester, and CLs from gene-targeted mice were used to show that LMP releases GrB in both a time- and concentration-dependent manner, and that the liberated GrB is responsible for cell death. The endogenous GrB inhibitor Serpinb9 (Sb9) protects CLs against LMP-induced death but is decreasingly effective as the extent of LMP increases. We also used these model stressors to show that GrB is the major effector of LMP-mediated death in T cells, but that in NK cells additional effectors are released, making GrB redundant. We found that limited LMP and GrB release occurs constitutively in proliferating lymphocytes and in NK cells engaged with targets in vitro. In Ectromelia virus-infected lymph nodes, working NK cells lacking Sb9 are more susceptible to GrB-mediated death. Taken together, these data show that a basal level of LMP occurs in proliferating and activated lymphocytes, and is increased on restimulation. LMP releases GrB from LROs into the lymphocyte cytoplasm and its ensuing interaction with Sb9 dictates whether or not the cell survives. The GrB-Sb9 nexus may therefore represent an additional mechanism of limiting lymphocyte lifespan and populations.     

The use of cyanate for the determination of NH 2 -terminal residues in N-acylated proteins

The results show that when the cyanate method is applied to proteins with certain N-acyl substituents, the terminal residues in their acylated form can appear in the hydantoin fraction and give rise to misleading conclusions on homogeneity. The survival of the N-acyl amino acid, and the loss of hydantoin, are governed by the conditions employed in the cyclization step. The blocked termini in N-acylated proteins can be demonstrated by using modified cyclization conditions.     

DNA extraction during giemsa differentiation of chromatids singly and doubly substituted with BrdU

Human lymphocytes were cultured in ³H-labelled BrdU. Cells were pretreated to induce differentiation, autoradiographed and Giemsastained. DNA extraction was deduced if grain counts were lower in differentiated mitoses compared with untreated controls. — The differentiation method involved sequential pretreatments with short wave UV and 2 × SSC at 60 ° C. This removed 34% of label from first division cells (with TB.TB chromosomes) but relatively more (53%) from second division (TB.BB chromosomes). In second division cells, about two thirds of label was lost from pale (BB) chromatids but only one third from dark (TB) chromatids. The UV and SSC pretreatments acted in collaboration, since neither alone reduced grain counts significantly. — On testing other methods, similar preferential DNA extraction was obtained with Perry and Wolff's FPG method, and with the hot salt pretreatment of Korenberg and Freedlender. However, good Giemsa differentiation could also be obtained using Hoechst 33258 and light pretreatments without any DNA loss. Reverse differentiation patterns (TB pale, BB dark) induced by warm acids resulted in extraction of nearly two thirds of ³H-BrdU label, but relative loss was the same from pale and dark chromatin. Direct reverse staining using alkaline Giemsa did not result in any loss of label. — Thus preferential DNA loss from pale stained chromatin underlies differentiation methods using light plus hot salt pretreatments, but it is not obligatory for good differentiation using other techniques.