Novel small molecule IL-6 inhibitor suppresses autoreactive Th17 development and promotes Treg development.

Multiple sclerosis (MS) is the leading cause of non-traumatic neurological disability in the United States in young adults, but current treatments are only partially effective, making it necessary to develop new, innovative therapeutic strategies. Myelin-specific interleukin (IL)-17-producing T helper type 17 (Th17) cells are a major subset of CD4 T effector cells (Teff ) that play a critical role in mediating the development and progression of MS and its mouse model, experimental autoimmune encephalomyelitis (EAE), while regulatory T cells (Treg ) CD4 T cells are beneficial for suppressing disease. The IL-6/signal transducer and activator of transcription 3 (STAT-3) signaling pathway is a key regulator of Th17 and Treg cells by promoting Th17 development and suppressing Treg development. Here we show that three novel small molecule IL-6 inhibitors, madindoline-5 (MDL-5), MDL-16 and MDL-101, significantly suppress IL-17 production in myelin-specific CD4 T cells in a dose-dependent manner in vitro. MDL-101 showed superior potency in suppressing IL-17 production compared to MDL-5 and MDL-16. Treatment of myelin-specific CD4 T cells with MDL-101 in vitro reduced their encephalitogenic potential following their subsequent adoptive transfer. Furthermore, MDL-101 significantly suppressed proliferation and IL-17 production of anti-CD3-activated effector/memory CD45RO+ CD4+ human CD4 T cells and promoted human Treg development. Together, these data demonstrate that these novel small molecule IL-6 inhibitors have the potential to shift the Teff  : Treg balance, which may provide a novel therapeutic strategy for ameliorating disease progression in MS.

Banff Histopathological Consensus Criteria for Preimplantation Kidney Biopsies.

The Banff working group on preimplantation biopsy was established to develop consensus criteria (best practice guidelines) for the interpretation of preimplantation kidney biopsies. Digitally scanned slides were used (i) to evaluate interobserver variability of histopathologic findings, comparing frozen sections with formalin-fixed, paraffin-embedded tissue of wedge and needle core biopsies, and (ii) to correlate consensus histopathologic findings with graft outcome in a cohort of biopsies from international medical centers. Intraclass correlations (ICCs) and univariable and multivariable statistical analyses were performed. Good to fair reproducibility was observed in semiquantitative scores for percentage of glomerulosclerosis, arterial intimal fibrosis and interstitial fibrosis on frozen wedge biopsies. Evaluation of frozen wedge and core biopsies was comparable for number of glomeruli, but needle biopsies showed worse ICCs for glomerulosclerosis, interstitial fibrosis and tubular atrophy. A consensus evaluation form is provided to help standardize the reporting of histopathologic lesions in donor biopsies. It should be recognized that histologic parameters may not correlate with graft outcome in studies based on organs deemed to be acceptable after careful clinical assessment. Significant limitations remain in the assessment of implantation biopsies.

The Banff 2015 Kidney Meeting Report: Current Challenges in Rejection Classification and Prospects for Adopting Molecular Pathology.

The XIII Banff meeting, held in conjunction the Canadian Society of Transplantation in Vancouver, Canada, reviewed the clinical impact of updates of C4d-negative antibody-mediated rejection (ABMR) from the 2013 meeting, reports from active Banff Working Groups, the relationships of donor-specific antibody tests (anti-HLA and non-HLA) with transplant histopathology, and questions of molecular transplant diagnostics. The use of transcriptome gene sets, their resultant diagnostic classifiers, or common key genes to supplement the diagnosis and classification of rejection requires further consensus agreement and validation in biopsies. Newly introduced concepts include the i-IFTA score, comprising inflammation within areas of fibrosis and atrophy and acceptance of transplant arteriolopathy within the descriptions of chronic active T cell-mediated rejection (TCMR) or chronic ABMR. The pattern of mixed TCMR and ABMR was increasingly recognized. This report also includes improved definitions of TCMR and ABMR in pancreas transplants with specification of vascular lesions and prospects for defining a vascularized composite allograft rejection classification. The goal of the Banff process is ongoing integration of advances in histologic, serologic, and molecular diagnostic techniques to produce a consensus-based reporting system that offers precise composite scores, accurate routine diagnostics, and applicability to next-generation clinical trials.

Splenic Irradiation for the Treatment of Severe Antibody-Mediated Rejection.

Patients requiring desensitization prior to renal transplantation are at risk for developing severe antibody-mediated rejection (AMR) refractory to treatment with plasmapheresis and intravenous immunoglobulin (PP/IVIg). We have previously reported success at graft salvage, long-term graft survival and protection against transplant glomerulopathy with the use of eculizumab and splenectomy in addition to PP/IVIg. Splenectomy may be an important component of this combination therapy and is itself associated with a marked reduction in donor-specific antibody (DSA) production. However, splenectomy represents a major operation, and some patients with severe AMR have comorbid conditions that substantially increase their risk of complications during and after surgery. In an effort to spare recipients the morbidity of a second operation, we used splenic irradiation in lieu of splenectomy in two incompatible live donor kidney transplant recipients with severe AMR in addition to PP/IVIg, rituximab and eculizumab. This novel approach to the treatment of severe AMR was associated with allograft salvage, excellent graft function and no short- or medium-term adverse effects of the radiation therapy. One-year surveillance biopsies did not show transplant glomerulopathy (tg) on light microscopy, but microcirculation inflammation and tg were present on electron microscopy.

Presentation and Outcomes of C4d-Negative Antibody-Mediated Rejection After Kidney Transplantation.

The updated Banff classification allows for the diagnosis of antibody-mediated rejection (AMR) in the absence of peritubular capillary C4d staining. Our objective was to quantify allograft loss risk in patients with consistently C4d-negative AMR (n = 51) compared with C4d-positive AMR patients (n = 156) and matched control subjects without AMR. All first-year posttransplant biopsy results from January 2004 through June 2014 were reviewed and correlated with the presence of donor-specific antibody (DSA). C4d-negative AMR patients were not different from C4d-positive AMR patients on any baseline characteristics, including immunologic risk factors (panel reactive antibody, prior transplant, HLA mismatch, donor type, DSA class, and anti-HLA/ABO-incompatibility). C4d-positive AMR patients were significantly more likely to have a clinical presentation (85.3% vs. 54.9%, p < 0.001), and those patients presented substantially earlier posttransplantation (median 14 [interquartile range 8-32] days vs. 46 [interquartile range 20-191], p < 0.001) and were three times more common (7.8% vs 2.5%). One- and 2-year post-AMR-defining biopsy graft survival in C4d-negative AMR patients was 93.4% and 90.2% versus 86.8% and 82.6% in C4d-positive AMR patients, respectively (p = 0.4). C4d-negative AMR was associated with a 2.56-fold (95% confidence interval, 1.08-6.05, p = 0.033) increased risk of graft loss compared with AMR-free matched controls. No clinical characteristics were identified that reliably distinguished C4d-negative from C4d-positive AMR. However, both phenotypes are associated with increased graft loss and thus warrant consideration for intervention.

Quantifying renal allograft loss following early antibody-mediated rejection.

Unlike antibody-mediated rejection (AMR) with clinical features, it remains unclear whether subclinical AMR should be treated, as its effect on allograft loss is unknown. It is also uncertain if AMR's effect is homogeneous across donor (deceased/live) and (HLA/ABO) antibody types. We compared 219 patients with AMR (77 subclinical, 142 clinical) to controls matched on HLA/ABO-compatibility, donor type, prior transplant, panel reactive antibody (PRA), age and year. One and 5-year graft survival in subclinical AMR was 95.9% and 75.7%, compared to 96.8% and 88.4% in matched controls (p = 0.0097). Subclinical AMR was independently associated with a 2.15-fold increased risk of graft loss (95% CI: 1.19-3.91; p = 0.012) compared to matched controls, but not different from clinical AMR (p = 0.13). Fifty three point two percent of subclinical AMR patients were treated with plasmapheresis within 3 days of their AMR-defining biopsy. Treated subclinical AMR patients had no difference in graft loss compared to matched controls (HR 1.73; 95% CI: 0.73-4.05; p = 0.21), but untreated subclinical AMR patients did (HR 3.34; 95% CI: 1.37-8.11; p = 0.008). AMR's effect on graft loss was heterogeneous when stratified by compatible deceased donor (HR = 4.73; 95% CI: 1.57-14.26; p = 0.006), HLA-incompatible deceased donor (HR = 2.39; 95% CI: 1.10-5.19; p = 0.028), compatible live donor (no AMR patients experienced graft loss), ABO-incompatible live donor (HR = 6.13; 95% CI: 0.55-67.70; p = 0.14) and HLA-incompatible live donor (HR = 6.29; 95% CI: 3.81-10.39; p < 0.001) transplant. Subclinical AMR substantially increases graft loss, and treatment seems warranted.

Banff 2013 meeting report: inclusion of c4d-negative antibody-mediated rejection and antibody-associated arterial lesions.

The 12th Banff Conference on Allograft Pathology was held in Comandatuba, Brazil, from August 19-23, 2013, and was preceded by a 2-day Latin American Symposium on Transplant Immunobiology and Immunopathology. The meeting was highlighted by the presentation of the findings of several working groups formed at the 2009 and 2011 Banff meetings to: (1) establish consensus criteria for diagnosing antibody-mediated rejection (ABMR) in the presence and absence of detectable C4d deposition; (2) develop consensus definitions and thresholds for glomerulitis (g score) and chronic glomerulopathy (cg score), associated with improved inter-observer agreement and correlation with clinical, molecular and serological data; (3) determine whether isolated lesions of intimal arteritis ("isolated v") represent acute rejection similar to intimal arteritis in the presence of tubulointerstitial inflammation; (4) compare different methodologies for evaluating interstitial fibrosis and for performing/evaluating implantation biopsies of renal allografts with regard to reproducibility and prediction of subsequent graft function; and (5) define clinically and prognostically significant morphologic criteria for subclassifying polyoma virus nephropathy. The key outcome of the 2013 conference is defining criteria for diagnosis of C4d-negative ABMR and respective modification of the Banff classification. In addition, three new Banff Working Groups were initiated.

Epidemiology, risk factors, and outcomes of Clostridium difficile infection in kidney transplant recipients.

BACKGROUND: We sought to describe the epidemiology and risk factors for Clostridium difficile infection (CDI) among kidney transplant recipients (KTR) between 1 January 2008 and 31 December 2010. METHODS: A single-institution retrospective study was conducted among all adult KTR with CDI, defined as a positive test for C. difficile by a cell cytotoxic assay for C. difficile toxin A or B or polymerase chain reaction test for toxigenic C. difficile. RESULTS: Among 603 kidney transplants performed between 1 January 2008 and 31 December 2010, 37 (6.1%) patients developed CDI: 12 (of 128; 9.4%) high-risk (blood group incompatible and/or anti-human leukocyte antigen donor-specific antibodies) vs. 25 (of 475; 5.3%, P = 0.08) standard-risk patients. The overall rate of CDI increased from 3.7% in 2008 to 9.4% in 2010 (P = 0.05). The median time to CDI diagnosis was 9 days, with 27 (73.0%) patients developing CDI within the first 30 days after their transplant, and 14 (51.8%) developing CDI within 7 days. A case-control analysis of 37 CDI cases and 74 matched controls demonstrated the following predictors for CDI among KTR: vancomycin-resistant Enterococcus colonization before transplant (odds ratio [OR]: 3.6, P = 0.03), receipt of an organ from Centers for Disease Control high-risk donor (OR: 5.9, P = 0.006), and administration of high-risk antibiotics within 30 days post transplant (OR: 6.6, P = 0.001). CONCLUSIONS: CDI remains a common early complication in KTR, with rates steadily increasing during the study period. Host and transplant-related factors and exposure to antibiotics appeared to significantly impact the risk for CDI among KTR.

Guidelines for the diagnosis of antibody-mediated rejection in pancreas allografts-updated Banff grading schema.

The first Banff proposal for the diagnosis of pancreas rejection (Am J Transplant 2008; 8: 237) dealt primarily with the diagnosis of acute T-cell-mediated rejection (ACMR), while only tentatively addressing issues pertaining to antibody-mediated rejection (AMR). This document presents comprehensive guidelines for the diagnosis of AMR, first proposed at the 10th Banff Conference on Allograft Pathology and refined by a broad-based multidisciplinary panel. Pancreatic AMR is best identified by a combination of serological and immunohistopathological findings consisting of (i) identification of circulating donor-specific antibodies, and histopathological data including (ii) morphological evidence of microvascular tissue injury and (iii) C4d staining in interacinar capillaries. Acute AMR is diagnosed conclusively if these three elements are present, whereas a diagnosis of suspicious for AMR is rendered if only two elements are identified. The identification of only one diagnostic element is not sufficient for the diagnosis of AMR but should prompt heightened clinical vigilance. AMR and ACMR may coexist, and should be recognized and graded independently. This proposal is based on our current knowledge of the pathogenesis of pancreas rejection and currently available tools for diagnosis. A systematized clinicopathological approach to AMR is essential for the development and assessment of much needed therapeutic interventions.

Rain, winds and haze during the Huygens probe's descent to Titan's surface.

The irreversible conversion of methane into higher hydrocarbons in Titan's stratosphere implies a surface or subsurface methane reservoir. Recent measurements from the cameras aboard the Cassini orbiter fail to see a global reservoir, but the methane and smog in Titan's atmosphere impedes the search for hydrocarbons on the surface. Here we report spectra and high-resolution images obtained by the Huygens Probe Descent Imager/Spectral Radiometer instrument in Titan's atmosphere. Although these images do not show liquid hydrocarbon pools on the surface, they do reveal the traces of once flowing liquid. Surprisingly like Earth, the brighter highland regions show complex systems draining into flat, dark lowlands. Images taken after landing are of a dry riverbed. The infrared reflectance spectrum measured for the surface is unlike any other in the Solar System; there is a red slope in the optical range that is consistent with an organic material such as tholins, and absorption from water ice is seen. However, a blue slope in the near-infrared suggests another, unknown constituent. The number density of haze particles increases by a factor of just a few from an altitude of 150 km to the surface, with no clear space below the tropopause. The methane relative humidity near the surface is 50 per cent.

Mls-1-like superantigen in the MA/MyJ mouse is encoded by a new mammary tumor provirus that is distinct from Mtv-7.

Mls-1 is an endogenous superantigen that leads to in vivo deletion and in vitro stimulation of T cell receptor (TCR) V beta 6-, 7-, 8.1-, and 9-expressing cells. The MA/MyJ mouse deletes the identical set of TCR from its mature T cell repertoire; however, it does not contain Mtv-7, the murine mammary tumor provirus (MMTV), whose sag gene encodes Mls-1. Interestingly, the superantigen activity of this mouse strain segregates with a new mammary tumor provirus, Mtv-43, not seen in other inbred strains. The predicted amino acid sequence of the sag gene of Mtv-43 was compared with that of Mtv-7. Strikingly, the COOH terminus of the two molecules is very similar, while all other MMTV-encoded superantigens differ 100% in this segment.

B cells are essential for murine mammary tumor virus transmission, but not for presentation of endogenous superantigens.

Murine mammary tumor viruses (MMTVs) are retroviruses that encode superantigens capable of stimulating T cells via superantigen-reactive T cell receptor V beta chains. MMTVs are transmitted to the suckling offspring through milk. Here we show that B cell-deficient mice foster nursed by virus-secreting mice do not transfer infectious MMTVs to their offspring. No MMTV proviruses could be detected in the spleen and mammary tissue of these mice, and no deletion of MMTV superantigen-reactive T cells occurred. By contrast, T cell deletion and positive selection due to endogenous MMTV superantigens occurred in B cell-deficient mice. We conclude that B cells are essential for the completion of the viral life cycle in vivo, but that endogenous MMTV superantigens can be presented by cell types other than B cells.

Banff '09 meeting report: antibody mediated graft deterioration and implementation of Banff working groups.

The 10th Banff Conference on Allograft Pathology was held in Banff, Canada from August 9 to 14, 2009. A total of 263 transplant clinicians, pathologists, surgeons, immunologists and researchers discussed several aspects of solid organ transplants with a special focus on antibody mediated graft injury. The willingness of the Banff process to adapt continuously in response to new research and improve potential weaknesses, led to the implementation of six working groups on the following areas: isolated v-lesion, fibrosis scoring, glomerular lesions, molecular pathology, polyomavirus nephropathy and quality assurance. Banff working groups will conduct multicenter trials to evaluate the clinical relevance, practical feasibility and reproducibility of potential changes to the Banff classification. There were also sessions on quality improvement in biopsy reading and utilization of virtual microscopy for maintaining competence in transplant biopsy interpretation. In addition, compelling molecular research data led to the discussion of incorporation of omics-technologies and discovery of new tissue markers with the goal of combining histopathology and molecular parameters within the Banff working classification in the near future.

The use of antibody to complement protein C5 for salvage treatment of severe antibody-mediated rejection.

Desensitized patients are at high risk of developing acute antibody-mediated rejection (AMR). In most cases, the rejection episodes are mild and respond to a short course of plasmapheresis (PP) / low-dose IVIg treatment. However, a subset of patients experience severe AMR associated with sudden onset oliguria. We previously described the utility of emergent splenectomy in rescuing allografts in patients with this type of severe AMR. However, not all patients are good candidates for splenectomy. Here we present a single case in which eculizumab, a complement protein C5 antibody that inhibits the formation of the membrane attack complex (MAC), was used combined with PP/IVIg to salvage a kidney undergoing severe AMR. We show a marked decrease in C5b-C9 (MAC) complex deposition in the kidney after the administration of eculizumab.

Subclinical rejection in stable positive crossmatch kidney transplant patients: incidence and correlations.

We reviewed 116 surveillance biopsies obtained approximately 1, 3, 6 and 12 months posttransplantation from 50 +XM live donor kidney transplant recipients to determine the frequency of subclinical cell-mediated rejection (CMR) and antibody-mediated rejection (AMR). Subclinical CMR was present in 39.7% of the biopsies at 1 month and >20% at all other time points. The presence of diffuse C4d on biopsies obtained at each time interval ranged from 20 to 30%. In every case, where histological and immunohistological findings were diagnostic for AMR, donor-specific antibody was found in the blood, challenging the long-held belief that low-level antibody could evade detection due to absorption on the graft. Among clinical factors, only recipient age was associated with subclinical CMR. Clinical factors associated with subclinical AMR were recipient age, positive cytotoxic crossmatch prior to desensitization and two mismatches of HLA DR 51, 52 and 53 alleles. Surveillance biopsies during the first year post-transplantation for these high-risk patients uncover clinically occult processes and phenotypes, which without intervention diminish allograft survival and function.

[Primary total elbow replacement for complex intra-articular distal humerus fractures]. / Primärer endoprothetischer Ellbogengelenkersatz bei komplexen intraartikulären distalen Humerusfrakturen.

INTRODUCTION: Open reduction and internal fixation of multifragmentary intra-articular fractures of the distal humerus often do not provide satisfactory results in elderly patients with osteoporosis. METHOD: From December 2001 to January 2008 a total elbow arthroplasty (Coonrad-Morrey, Zimmer, USA) was performed on 12 patients (average age 81+/-9 ears) who presented with a type C distal humeral fracture. The mean time of follow-up with clinical and radiological assessment was 28+/-17 months. RESULTS: The Mayo score showed a good functional result with an average of 81+/-9 out of 100. DASH and SECEC scores showed a fair result with respect to elbow function (43+/-8 and 68+/-7 points, respectively). The average range of motion of all patients was 120-33-0 degrees. Heterotopic ossifications were found by X-ray examination in 4 cases and asymptomatic radiolucent lines in 4 cases. CONCLUSION: Primary total elbow arthroplasty for complex intra-articular distal humerus fractures in elderly patients has good functional results and is an alternative to osteosynthesis.

Banff 07 classification of renal allograft pathology: updates and future directions.

The 9th Banff Conference on Allograft Pathology was held in La Coruna, Spain on June 23-29, 2007. A total of 235 pathologists, clinicians and scientists met to address unsolved issues in transplantation and adapt the Banff schema for renal allograft rejection in response to emerging data and technologies. The outcome of the consensus discussions on renal pathology is provided in this article. Major updates from the 2007 Banff Conference were: inclusion of peritubular capillaritis grading, C4d scoring, interpretation of C4d deposition without morphological evidence of active rejection, application of the Banff criteria to zero-time and protocol biopsies and introduction of a new scoring for total interstitial inflammation (ti-score). In addition, emerging research data led to the establishment of collaborative working groups addressing issues like isolated 'v' lesion and incorporation of omics-technologies, paving the way for future combination of graft biopsy and molecular parameters within the Banff process.

Transforming growth factor alpha protection against drug-induced injury to the rat gastric mucosa in vivo.

This study was designed to determine whether transforming growth factor alpha (TGF alpha) protects rat gastric mucosa against ethanol- and aspirin-induced injury. Systemic administration of TGF alpha dose-dependently decreased 100% ethanol-induced gastric mucosal injury; a dose of 50 micrograms/kg delivered intraperitoneally 15 min before ethanol decreased macroscopic mucosal injury by > 90%. At the microscopic level, TGF alpha prevented deep gastric necrotic lesions and reduced disruption of surface epithelium. Pretreatment with orogastric TGF alpha (200 micrograms/kg) only partially (40%) decreased macroscopic ethanol damage. Intraperitoneal administration of TGF alpha at a dose of 10 micrograms/kg, which does not significantly inhibit gastric acid secretion, decreased aspirin-induced macroscopic damage by > 80%. TGF alpha protection does not seem to be mediated by prostaglandin, glutathione, or ornithine decarboxylase-related events, as evidenced by lack of influence of the inhibition of their production. Pretreatment with the sulfhydryl blocking agent N-ethylmaleimide partially abolished (40%) the protective effect of TGF alpha. In addition, systemic administration of TGF alpha resulted in a two-fold increase in tyrosine phosphorylation of phospholipase C-gamma 1 and in a time- and dose-dependent increase in levels of immunoreactive insoluble gastric mucin; these events occurred in a time frame consistent with their participation in the protective effect of TGF alpha.

Quantitative detection of promoter hypermethylation as a biomarker of acute kidney injury during transplantation.

Aberrant promoter hypermethylation, also known as epigenetics, is thought to be a promising biomarker approach to diagnose malignancies. Kidney repair after injury is a recapitulation of normal morphogenesis, with similarities to malignant transformation. We hypothesized that changes in urine epigenetics could be a biomarker approach during early kidney transplant injury and repair. We examined urine DNA for aberrant methylation of two gene promoters (DAPK and CALCA) by quantitative methylation-specific polymerase chain reaction from 13 deceased and 10 living donor kidney transplant recipients on postoperative day 2 and 65 healthy controls. Results were compared with clinical outcomes and to results of the kidney biopsy. Transplant recipients were significantly more likely to have aberrant hypermethylation of the CALCA gene promoter in urine than healthy controls (100% vs 31%; P < .0001). There was increased CALCA hypermethylation in the urine of deceased versus living donor transplants (21.60 +/- 12.5 vs 12.19 +/- 4.7; P = .04). Furthermore, there was a trend toward increased aberrant hypermethylation of urine CALCA in patients with biopsy-proven acute tubular necrosis versus acute rejection and slow or prompt graft function (mean: 20.40 +/- 6.9, 13.87 +/- 6.49, 17.17 +/- 13.4; P = .67). However, there was no difference of CALCA hypermethylation in urine of patients with delayed graft function versus those with slow or prompt graft function (16.9 +/- 6.2 vs 18.5 +/- 13.7, respectively; P = .5). There was no aberrant hypermethylation of DAPK in the urine of transplant patients. Urine epigenetics is a promising biomarker approach for acute ischemic injury in transplantation that merits future study.

Analysis of genomic instability in Li-Fraumeni fibroblasts with germline p53 mutations.

Germline p53 mutations are frequently observed in the normal DNA of cancer-prone patients with Li-Fraumeni syndrome (LFS). Fibroblasts from LFS patients develop chromosomal aberrations, loss of cell cycle control, and spontaneous immortalization. We transfected four different mutant p53 genes into human skin fibroblasts from normal donors with two copies of wild-type p53 (p53(wt/wt)). Each mutant p53 expression-plasmid induced genomic instability equivalent to that seen in LFS cells. To test the role of wild-type and mutant p53 alleles in DNA replication and fidelity in LFS cells, we analysed the replication of the SV40-based shuttle vector pZ189 in four types of cells. We used p53(wt/mut) and p53(mut/-) LFS fibroblasts, and p53(-/-) non-LFS cells. Replication of pZ189 in vivo was significantly reduced by the presence of a p53(wt) allele. To show that this was not just due to inhibition of the function of T-antigen in SV40-based replication, we constructed a shuttle vector, pZ402, that contains a mutation in SV40 T-antigen which blocks its ability to interact with p53. Replication of pZ402 in LFS cells was also reduced by the presence of p53(wt), indicating that p53 can inhibit replication by interacting with proteins within the cellular replication machinery. Replicative errors in this shuttle vector are detected as mutations in a marker gene, supF. In addition to supF mutations, we observed deletion of a portion of the SV40 T-antigen gene in 100% of replicated plasmid pZ189 mutants (supF-) from the p53(wt/mut) fibroblasts and in 88% of the supF mutants from the p53(mut/-) (amino acid 175 arg to his) LFS cells. In one cell strain of immortal LFS cells, P53(mut/-) , containing a p53 frameshift mutation at amino acid 184, pZ189 replication yielded very few of these deleted shuttle vector plasmids (15%). These large deletions were not detected in plasmids replicated in p53(-/-) non-LFS cells, Saos-2 cells. Replicated plasmids with a normal supF gene were never found to have this large deletion regardless of the cell from which they were derived. Because the supF gene is not in the same region of the shuttle vector as the T-antigen gene it appears that second, independent gene deletions are frequent when replicative errors in supF occur in cells with a mutant p53. We conclude, therefore, that p53(wt/mut) LFS cells contain an activity that promotes mutations. Such an activity, which is likely to be due to the p53(mut), could result in the high rate of chromosomal instability and allelic loss of the wild-type p53 observed as these cells spontaneously immortalize.