Background: The tumor suppressor gene p53 is expressed without apoptosis in the limbal basal stem cells of all pterygia and limbal tumors and most pingueculae from which these growths seem to originate. Oncogenic human papillomaviruses (HPVs) have been found in pterygia and limbal tumors, and HPV and p53 overexpression commonly coexist in oropharyngeal and penile carcinomas. Objective: To search for HPV DNA as a cofactor in the development of pingueculae, pterygia, and limbal tumors. Methods: We examined specimens-1 of pinguecula, 13 of pterygia (7 primary, 1 recurrent, 1 with dysplasia, and 4 primary not tested for p53), and 10 of limbal tumors (2 with actinic keratosis dysplasia, 1 with conjunctival intraepithelial neoplasia, 3 with carcinoma in situ, and 4 with squamous cell carcinoma)-expressing p53. Specimens were tested for the presence of HPV DNA by the polymerase chain reaction using degenerate consensus primers for the highly conserved portion of the L1 region that encodes a capsid protein of the virus. This assay has a wide spectrum with capability of detecting essentially all known HPV types. Nested polymerase chain reaction was performed on all specimens. Primers of the CLINICAL SCIENCES

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We report here a new mechanism for allelic discrimination-allele-specific Holliday Junction formation. The Holliday Junction (HJ) is a unique DNA structure that can be formed in a sequence-nonspecific manner by routine PCR. To cause the PCR-based HJ formation to occur in an allele-specific manner, the PCR primers are manipulated such that an extra mismatch next to a SNP of interest is introduced between a target and a reference amplicon and a GC-clamp is added. Based on this new mechanism, novel SNP genotyping methods were developed, including a homogeneous fluorescence polarization (FP) competition assay that requires neither labeled primers/probes nor expensive enzymes/substrates. Using this novel genotyping technology, we were able to convert >95% of SNP sequences into genotyping assays that work well under a universal set of assay conditions and achieved 100% accuracy in clinical samples.SNPs can be detected in either a sequence-specific or sequence-nonspecific way (Kowk 2001). Sequence-specific detection relies on four general mechanisms for allelic discrimination: allele-specific hybridization, allele-specific nucleotide incorporation, allele-specific oligonucleotide ligation, and allele-specific invasive cleavage (Kowk 2001). We describe here a fifth mechanism for allelic discrimination: allele-specific Holliday Junction (HJ) formation.It has been shown by Panyutin and Hsieh (1993) that two partial duplexes will quickly form a four-stranded cruciform Holliday Junction if their tails are complementary enough to anneal to each other (Fig. 1A). The two partial duplexes that form the Holliday Junction will then undergo strand exchange through spontaneous branch migration, during which the energy loss due to the breaking of a base pair is coupled with the energy gain from the formation of a new base pair (Panyutin and Hsieh 1993). The strand exchange will go to completion and the Holliday Junction will quickly resolve into two duplexes when the sequences of the duplex portions of the two partial duplexes are the same (Panyutin and Hsieh 1993). However, when there is a difference between the duplex portions of the two partial duplexes, in the presence of Mg 2+ , the spontaneous branch migration will encounter an energy barrier at the mismatch position, leading to the formation of stable Holliday Junctions (Panyutin and Hsieh 1993). Based on Panyutin's discovery, Lishanski and coworkers developed a method for sequence-nonspecific mutation detection using PCR-amplified DNA fragments ( Fig. 1B; Lishanski 2000; Lishanski et al. 2000). Like hybridization or heteroduplex formation, Holliday Junction formation is itself sequence-nonspecific. To make Holliday Junctions form in an allele-specific manner, it is necessary to make the target amplicons and the reference amplicons short enough so that the possibility for the existence of an additional SNP is negligibly small. However, when the forward and the reverse primers are separated only by one or a few bases, a single-base mismatch can no longer impede branch migratio...

Non-Hodgkin's lymphoma (NHL) occurring as a synchronous malignancy with chronic myelogenous leukemia (CML) is rare. To our knowledge, this is the first case reported of a patient who developed mantle cell lymphoma (MCL) after therapy with imatinib mesylate for CML. After a 3-year history of CML, the patient developed a lymphocytosis associated with diarrhea, anorexia, and weight loss. Imaging studies revealed abdominal adenopathy and extensive lymphomatous infiltration of the liver, stomach, pancreas, and kidneys. Flow cytometric and cytogenetic studies were consistent with MCL. Fluorescence in situ hybridization (FISH) of the bone marrow revealed a genetically distinct lymphoid neoplasm rather than an extramedullary blast crisis of CML. The development of lung cancer, prostate cancer, CML and MCL in this patient suggests a genetic predisposition, although other factors, including environmental exposures and therapy with imatinib mesylate could have had a contributory or synergistic role in the development of MCL. Am.

F resh osteochondral allografts have been widely used to treat cartilage lesions for more than 100 years 1 . Transplantation of cartilage and bone in the form of an allograft allows osseous healing while maintaining the articular cartilage architecture. This composite tissue transplant remains intact in vivo for extensive periods of time with a favorable mechanical and biological environment. The chondrocytes of the graft are thought to actively remodel the extracellular matrix environment, and thus contribute to the tissue integrity. We recently reported that allograft cells could survive up to twenty-nine years after transplantation without the need for systemic immunosuppression 2 . Although mosaic cell populations have been demonstrated in other forms of transplantation, these have always been under the umbrella of long-term systemic immunosuppression 3,4 . In a classic study, Langer and Gross 5 showed that intact articular cartilage surfaces obtained by removing the subchondral bone of rat femoral heads and filling of the osseous segments with acrylic cement exhibited essentially no humoral immune response in contrast to that seen with minced cartilage or isolated chondrocyte transplants. This finding has been attributed to the so-called ''immunoprivileged'' status of articular cartilage, which protects the chondrocytes from the immune system of the host.The extent to which allograft chondrocytes retain their gene expression profiles and chondrogenic capacities remains unknown. Our goal was to compare gene expression, proliferation rate, and chondrogenic potential between host and allograft chondrocytes isolated three years after an unsuccessful fresh osteochondral allograft transplant in the knee. The patient was informed that data concerning her case would be submitted for publication, and she provided consent. The study was performed in full compliance with our institutional review board (IRB). Our IRB at University of California Davis Medical Center does not consider case reports to be research and does not expressly provide IRB approval. We have sought IRB approval for case reports on a number of occasions in the past and have been given written documentation of this policy. Case Report Tissue SourceA forty-eight-year-old woman with early osteoarthritis of the knee was treated with a fresh osteochondral allograft with use of cylindrical plugs prepared with commercially available instrumentation (Arthrex, Naples, Florida); the plugs were applied to the trochlea (20 · 20-mm plug), the medial femoral condyle (20 · 20-mm plug), and the lateral femoral condyle (15 · 15-mm plug). The graft was a whole fresh distal part of a femur (University of Miami Tissue Bank, Miami, Florida) from an eighteen-year-old male donor implanted twelve days after harvest. The tissue bank provided the allografts submerged in culture media (RPMI-1640 or lactated Ringer solution) with antibiotics at a refrigerated temperature (1°C to 10°C, never frozen). Transplants approximately 1.0 to 1.5 cm in diameter were created from the regio...