Differential Effect of Diarrhea on FK506 Versus Cyclosporine A Trough Levels and Resultant Prevention of Allograft Rejection in Renal Transplant Recipients

Diarrhea is the most frequently reported adverse event in patients treated with mycophenolate mofetil. Twenty-six renal transplant patients on a mycophenolate mofetil-based immunosuppressive regime with persistent afebrile diarrhea were examined. Diarrhea caused a significant rise in FK-506 trough levels despite intake of stable doses, necessitating FK-506 dose reductions of 30% to obtain pre-diarrhea trough levels. In contrast, trough levels of cyclosporine A remained stable without dose adjustments. This suggests that absorption and/or metabolism is differentially altered for FK506 compared with cyclosporine A in patients with diarrhea. In nine patients mycophenolate mofetil was reduced or stopped because of persistent diarrhea without identifiable cause. This resulted in end-stage renal disease because of chronic rejection in two patients, and in acute rejection in two patients, all taking FK506 and steroids. Therefore, dose adjustments of FK506 in patients with diarrhea must be carefully monitored, especially when doses of mycophenolate mofetil are also reduced.     

Characterization of RAFTK, a novel focal adhesion kinase, and its integrin-dependent phosphorylation and activation in megakaryocytes

We have recently isolated a cDNA encoding a novel human intracellular tyrosine kinase, termed RAFTK (for a related adhesion focal tyrosine kinase). The RAFTK cDNA, which encodes a polypeptide of 1,009 amino acids, shares 65% homology to the focal adhesion kinase (FAK), including several consensus motifs. In this report, we describe the biochemical characterization and functional analysis of the RAFTK protein. Coexpression of RAFTK and FAK proteins in megakaryocytic cells and blood platelets was observed. Using a specific antibody to RAFTK and the monoclonal antibody 2A7 to FAK, FAK and RAFTK could be distinguished antigenically. RAFTK had intrinsic tyrosine kinase and autokinase activities. It was phosphorylated on tyrosine in growing cultures of COS cells transfected with the pCDNAIII/flag-RAFTK expression vector containing the RAFTK cDNA ligated with the 8 amino acid flag peptide sequence. Similar to FAK, dephosphorylation of RAFTK was observed when adherent transfected COS cells were detached. Phosphorylation was regained upon replating of these cells on the fibronectincoated dishes. Analysis of tyrosine-phosphorylated RAFTK from adherent transfected COS cells showed that the Src homology 2 (SH2) domains of the Src and Fyn protein kinases as well as the Grb2 adaptor protein were able to specifically associate with RAFTK. Tyrosine phosphorylation of endogenous RAFTK was observed upon fibronectin-induced activation of human megakaryocytic cells. Furthermore, colocalization of RAFTK protein with vinculin, a focal adhesion protein, was observed by confocal microscopy in focal adhesion- like structures in adherent CMK cells and in transfected pCDNAIII/flag- RAFTK COS cells upon fibronectin activation. These data suggest that RAFTK is a novel member of the FAK family, that it localizes to focal adhesion-like structures in CMK megakaryocytic cells, that it participates in integrinmediated signaling pathways in megakaryocytes, and that it is able to associate with the tyrosine kinases Src and Fyn as well as the adaptor protein Grb2 via SH2-phosphotyrosine interactions.     

CD40 is a prognostic marker in primary cutaneous malignant melanoma.

CD40 is a receptor at the surface of B lymphocytes with important functions in the immune response. CD40 has also been found on a variety of carcinoma and melanoma cell lines where it has been suggested to serve as a possible receptor for mitogenic signals. We studied the expression and distribution of CD40 in paraffin sections of 71 uniformly treated malignant melanomas (MMs) with a long clinical follow-up using well known monoclonal antibodies. For comparison, 71 benign nevi were also studied. Common acquired nevi occasionally expressed CD40 in nests or single cells at the dermo-epidermal junction; no immunoreactivity was observed in the dermal part of acquired nevi, and all Spitz' nevi were entirely negative. One-third of large congenital nevi expressed CD40 in small clusters of heavily pigmented, epithelioid cells, corresponding to so-called proliferative nodules. In 41 of 71 MMs, CD40 was expressed in single or clustered neoplastic melanocytes; 9 cases showed CD40 expression only in the radial growth phase, and in 32 cases, the vertical growth phase showed CD40 expression. The same staining pattern was obtained with other anti-CD40 monoclonal antibodies, directed to different epitopes of the CD40 molecule. In 29 of 32 MMs showing CD40 in the vertical growth phase, expression of the CD40 ligand (CD40L) was studied; in 13 of these 29, CD40L was found in the same tumor areas that expressed CD40. Analysis of 28 metastases from 24 MM patients showed in the majority of cases a similar, scattered or nodular staining pattern as observed in the primary tumor. Patients expressing CD40 in the vertical growth phase of their MM did not differ significantly from CD40-negative patients with respect to any of the known prognostic parameters but showed a significantly shorter tumor-free survival. Patients with CD40+ CD40L+ MM tended to have a shorter tumor-free survival than those lacking CD40L. We conclude that CD40 represents a novel prognostic parameter in primary cutaneous MM. The co-localization of CD40 and CD40L suggests an autocrine growth loop in the vertical growth phase of MM.     

STXBP1: the second synaptic vesicle release gene involved in epilepsy

De novo mutations in the gene encoding STXBP1 (MUNC18‐1) cause early infantile epileptic encephalopathy
Saitsu et al. (2008)
Nature Genetics 40: 782–788