Activation-induced cell death (AICD) as well as programmed cell death (PCD) serve to control the expansion of activated T cells to limit untoward side effects of continued effector responses by T cells and to maintain homeostasis. AICD of T cells in tumor immunotherapy can be counterproductive particularly if the activated T cells undergo apoptotic death after the very first secondary encounter of the specific epitope. We examined the extent to which tumor epitope-specific CTLs that are activated and expanded in an in vitro-matured dendritic cell-based primary stimulation protocol undergo AICD following their first secondary encounter of the cognate epitope. Using the MART-127–35 epitope as a prototype vaccine epitope, we also examined whether these CTLs could be rescued from AICD. Our results demonstrate that a substantial fraction of MART-127–35 epitope-specific primary CTLs undergo AICD upon the very first secondary encounter of the cognate epitope. The AICD in these CTLs is neither caspase dependent nor is it triggered by the extrinsic death signaling pathways (Fas, TNFR, etc.). These CTLs, interestingly, could be rescued from AICD by the JNK inhibitor, SP600125. We also found that SP600125 interferes with their IFN-γ response but does not block their cytolytic function. The rescued CTLs, however, regain their capacity to synthesize IFN-γ if continued in culture without the inhibitor. These observations have implications in tumor immunotherapy and in further studies for regulation of AICD in CTLs.

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A retrospective evaluation of 200 consecutive recipients of autologous peripheral blood stem cell transplantation (PBSCT) was conducted to ascertain the incidence and outcome of infection with Clostridium difficile. The diagnosis was confirmed in 14 patients with diarrhea (15 episodes) at a median of 33 days after stem cell infusion. Five patients were neutropenic at the time of diagnosis. Every individual had adverse known risk factors such as recent or current use of antibiotic, corticosteroid and antiviral therapy, recent administration of myeloablative chemotherapy and numerous, prolonged periods of hospitalization. Diarrhea, frequently hemorrhagic, was the most common presenting feature along with fever, abdominal cramps and abdominal distention. Diagnosis was established by the stool-cytotoxin test. Response to standard treatment with oral vancomycin or metronidazole was prompt despite the presence of several adverse prognostic features in these patients. There was only one instance of relapse which was also treated successfully. Several transplant-related variables such as age, sex, underlying malignancy, myelo-ablative regimen, duration of neutropenia, and prophylactic use of oral ampicillin underwent statistical analysis but failed to be predictive of C. difficile infection in such a setting. Finally, C. difficile is not uncommon after autologous PBSCT and must be included in the differential diagnosis in any such patient with diarrhea.

We have constructed a transformation-defective polyoma virus mutant (Py 1387-T) that directs the synthesis of a normal small tumor antigen, a functional large tumor antigen, and a truncated (51,000-dalton) middle-sized tumor (mT) antigen that lacks 37 amino acids at its COOH terminus. The shortened mT polypeptide is missing the hydrophobic "tail" thought to be responsible for the anchorage of this protein into the plasma membrane and is in fact in cytosol fractions. This truncated mT polypeptide is inactive in an in vitro protein kinase assay and is altered in its phosphorylation in viva Mutant 1387-T differs from wildtype virus in having a T-A base pair instead of a C-G base pair at nucleotide position 1387. This change was introduced into viral DNA by using a synthetic undecanucleotide as a specific mutagen. Wild-type polyoma DNA was rendered single stranded by molecular cloning into coliphage M13. The oligonucleotide, which hybridizes with a mismatch at the site to be altered, was used to prime the synthesis ofdouble-stranded closed circular DNA. Progeny recombinant phages were screened by DNA sequence analysis for the desired base change. The polyoma mutant was reconstructed from recombinant phage replicative form DNA molecules containing the mutation.The early region of polyoma virus encodes three proteins, the small [22-kilodalton (kDal)], the middle-sized (56-kDal), and the large (100-kDal) tumor (T) antigens. The large T antigen is required for transformation by intact virus (1-3) and may function by promoting stable association ofthe viral genome with cellularDNA. An intact large T protein is not essential for maintenance ofthe transformed phenotype (4)(5)(6)(7)(8)(9). Small T and middle T (mT) antigens have been implicated in maintenance of the transformed state and in tumorigenicity by the virus. The nontransforming hr-t mutants (10-12) have lesions affecting both the small T and mT polypeptides but not large T protein (4,(13)(14)(15).The importance of the mT polypeptide in transformation has been demonstrated directly; DNA coding only for mT antigen can transform rat cells (16). Mutants in which mT, but not small T, antigen is altered are affected in their transformation properties (17-19). When deletions affecting the COOH-terminal region of mT antigen are created in cloned polyoma DNA, the ability of that DNA to transform cells is reduced (20).Phosphorylation is important to mT antigen function. A protein kinase activity that phosphorylates tyrosine residue 315 of mT antigen is present in T-antigen immunoprecipitates (21-25). Middle T antigen can be resolved into two species, 56 kDal and 58 kDal, that differ strikingly in activity in the in vitro kinase reaction (24,25). These species also differ in their patterns of phosphorylation in vivo. The high specific activity 58-kDal form is phosphorylated in vivo at different serine or threonine residues than is the 56-kDal form (24, 25). These results suggest that the activity of mT antigen may be regulated by cellular protein kinase(s).mT antig...

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