Immunolocalization of lacrimal gland PKC isoforms. Effect of phorbol esters and cholinergic agonists on their cellular distribution.

In previous studies, we showed that lacrimal gland acini express three isoforms of protein kinase C (PKC): PKCalpha,-delta, and -epsilon. In the present study, we report the identification of two other PKC isoforms, namely PKCmu and -iota/lambda. Using immunofluorescence techniques, we showed that these isoforms are differentially located. PKCalpha and -mu showed the most prominent membrane localization, whereas PKCdelta, -epsilon and -iota/lambda were mainly cytosolic. Using cell fractionation and western blotting techniques, we showed that the phorbol ester, phorbol 12,13-dibutyrate (PdBu, 10(-6) M), translocated all PKC isoforms, except PKCiota/lambda, from the soluble fraction into the particulate fraction. The effect was maximum at 5 min and persisted at 10 min. PKCepsilon was the most responsive to PdBu reaching almost maximal translocation at a PdBu concentration as low as 10(-9) M. The cholinergic agonist, carbachol (10(-5) and 10(-3) M), induced translocation which was transient for PKCdelta, and -mu, but persisted for 10 min for PKCepsilon. Carbachol did not translocate PKCalpha and, like PdBu, did not translocate PKCiota/lambda. We concluded that lacrimal gland PKC isoforms are differentially localized and that they translocate differentially in response to phorbol esters and cholinergic agonists.

Mucocutaneous autoimmune syndrome following fludarabine therapy for low-grade non-Hodgkin's lymphoma of B-cell type (B-NHL).

A 40-year-old patient with low-grade B-NHL developed a generalized macular-papular rash following the first cycle of fludarabine treatment which progressed to a complete epidermal necrolysis following the second cycle. Clinical symptoms and the results of the direct and indirect immunofluorescence were consistent with a mucocutaneous autoimmune syndrome (pemphigus). Immunohistochemical analysis demonstrated a dense epidermal infiltration of CD8+ lymphocytes associated with the histological features of single-cell necrosis of keratinocytes. Early and aggressive immunosuppressive treatment with steroids, cyclophosphamide, and high-dose immunoglobulins resulted in regression of symptoms and complete reconstitution of epidermal integrity. The malignant lymphoma has completely regressed. The findings suggest a fludarabine-induced defect in immunosurveillance--resulting in the uncontrolled activation of autoaggressive T-cell clones--as a pathogenetic mechanism of this life-threatening dermatological complication.

The utilization of individual VH exons in the primary repertoire of adult BALB/c mice.

The utilization of VH gene families is severely biased during fetal development, such that D-region proximal VH genes are overrepresented. After birth the VH repertoire becomes more equally representative of the total inherited set of functional VH genes. To investigate the extent of this normalization process, we have determined the relative utilization of individual VH exons in the pre-immune repertoire of adult BALB/c spleen cells. Large samples of IgM heavy chain transcripts from polyclonally activated B cells were captured in a cDNA phage library and screened by hybridization using highly specific oligonucleotide probes. These studies revealed that the utilization of particular VH exons can differ by an order of magnitude. Significantly, the VH genes most under-represented in the pre-immune repertoire are located in the region of the Igh locus most distal to the DjhCh region. We suggest that the chromosomal position of a particular VH gene may influence its utilization and that the normalization of the adult repertoire is incomplete.