Ex vivo staining of metastatic lymph nodes by class I major histocompatibility complex tetramers reveals high numbers of antigen-experienced tumor-specific cytolytic T lymphocytes

Characterization of cytolytic T lymphocyte (CTL) responses to tumor antigens has been impeded by a lack of direct assays of CTL activity. We have synthesized reagents ("tetramers") that specifically stain CTLs recognizing melanoma antigens. Tetramer staining of tumor-infiltrated lymph nodes ex vivo revealed high frequencies of tumor-specific CTLs which were antigen-experienced by surface phenotype. In vitro culture of lymph node cells with cytokines resulted in very large expansions of tumor-specific CTLs that were dependent on the presence of tumor cells in the lymph nodes. Tetramer-guided sorting by flow cytometer allowed isolation of melanoma-specific CTLs and confirmation of their specificity and their ability to lyse autologous tumor cells. Our results demonstrate the value of these novel reagents for monitoring tumor-specific CTL responses and for generating CTLs for adoptive immunotherapy. These data also indicate that strong CTL responses to melanoma often occur in vivo, and that the reactive CTLs have substantial proliferative and tumoricidal potential.     

Isolation and characterization of a monoclonal anti-quadruplex DNA antibody from autoimmune "viable motheaten" mice

A cell line that produces an autoantibody specific for DNA quadruplex structures has been isolated and cloned from a hybridoma library derived from 3-month-old nonimmunized autoimmune, immunodeficient "viable motheaten" mice. This antibody has been tested extensively in vitro and found to bind specifically to DNA quadruplex structures formed by two biologically relevant sequence motifs. Scatchard and nonlinear regression analyses using both one- and two-site models were used to derive association constants for the antibody-DNA binding reactions. In both cases, quadruplexes had higher association constants than triplex and duplex molecules. The anti-quadruplex antibody binds to the quadruplex formed by the promoter-region-derived oligonucleotide d(CGCG4GCG) (Ka = 3.3 x 10(6) M-1), and has enhanced affinity for telomere-derived quadruplexes formed by the oligonucleotides d(TG4) and d(T2G4T2G4T2G4T2G4) (Ka = 5.38 x 10(6) and 1.66 x 10(7) M-1, respectively). The antibody binds both types of quadruplexes but has preferential affinity for the parallel four-stranded structure. In vitro radioimmunofilter binding experiments demonstrated that purified anti-DNA quadruplex antibodies from anti-quadruplex antibody-producing tissue culture supernatants have at least 10-fold higher affinity for quadruplexes than for triplex and duplex DNA structures of similar base composition and length. The antibody binds intramolecular DNA triplexes formed by d(G4T3G4T3C4) and d(C4T3G4T3G4), and the duplex d(CGCGCGCGCG)2 with an affinities of 6. 76 x 10(5), 5.59 x 10(5), and 8.26 x 10(5) M-1, respectively. Competition experiments showed that melted quadruplexes are not effective competitors for antibody binding when compared to native structures, confirming that the quadruplex is bound structure-specifically. To our knowledge, this is the first immunological reagent known to specifically recognize quadruplex structures. Subsequent sequence analysis demonstrates homologies between the antibody complementarity determining regions and sequences from Myb family telomere binding proteins, which are hypothesized to control cell aging via telomeric DNA interactions. The presence of this antibody in the autoimmune repertoire suggests a possible linkage between autoimmunity, telomeric DNA binding proteins, and aging.     

Hepatectomy with hypothermic perfusion of the liver

Whereas most liver resections can be performed within 60 min, the period of vascular clamping and resulting ischemia may prove too short to allow complex major liver resections (MLR) especially on diseased livers. To overcome this problem, cooling of the liver with 4 degrees C preservations solution routinely used in liver transplantation may be used in three different approaches to MLR: I "In situ": the liver remains in the abdomen and integrity of afferent and efferent vessels is conserved. II "Ex situ-in vivo": the liver exteriorized from the abdomen by transecting all hepatic veins, remains connected to the porta hepatis. III "Ex vivo": the liver being removed from the abdomen, the MLR is performed extracorporeally. Of 15 MLR reported here, 11 were performed "in situ" and 4 "ex situ-in vivo"/Nowadays, the liver surgeon's "toolbox" must contain hypothermic liver perfusion. In carefully selected cases, these techniques allow MLR on diseases livers or mandating complex vascular procedures.     

Anterior cervical corpectomy for cervical spondylotic myelopathy

OBJECTIVE: To evaluate the efficacy of anterior surgery for the treatment of cervical spondylotic myelopathy, we have reviewed our experience with anterior cervical corpectomy (ACC) at the University of Florida, specifically analyzing neurological outcomes and complications. These results have been compared with historical control subjects receiving laminectomy or "no treatment." METHODS: Between 1982 and 1992, 93 ACC operations were performed for the primary diagnosis of cervical spondylotic myelopathy. This consecutive series of patients was reviewed retrospectively. Age, gender, pre- and postoperative myelopathy severity, number of levels decompressed, and neurological complications were assessed. Myelopathy severity was graded using the Nurick myelopathy grading system. The average follow-up period was 39 months (range, 2-137 mo). RESULTS: Symptomatic improvement was achieved for 92% of patients (F = 28.9, df = 2172, P < 0.001). Nurick scores reflected improvement for 86% of patients, with the conditions of 13% remaining unchanged and only one patient showing worsening. Preoperative myelopathy severity was weakly correlated with age (P < 0.05) but was not correlated with gender or number of levels decompressed. Similarly, postoperative myelopathy severity was not significantly correlated with age, gender, preoperative myelopathy severity, or number of levels decompressed. ACC-treated patients showed an average improvement of 1.24 points on the Nurick scale, compared with an improvement of 0.07 points for patients treated with laminectomy (P < 0.001) and a deterioration of 0.23 points for patients undergoing conservative treatment (P < 0.001). Complications were slightly more likely to occur in older patients (P < 0.05). The number of levels decompressed was not significantly correlated with complications. Only one permanent neurological complication was seen in this series of patients. CONCLUSION: We conclude that ACC is a safe and effective treatment for cervical spondylotic myelopathy. In an average of 39 months, ACC showed improved results in terms of myelopathy scores, compared with historical control subjects receiving either no treatment or laminectomy. Age, gender, preoperative myelopathy severity, and extent of disease were not negative predictors of clinical outcomes.     

Visuospatial neglect: the ultimate deconstruction?

Unilateral visuospatial neglect is now widely acknowledged to be a highly heterogeneous condition: The overt manifestations of visual neglect can vary as a function of task, spatial domain, and mode of response (at least). Double dissociations (sometimes of the strong form) have already been reported between most of the components of what was originally thought to be a relatively stable construct within the visual modality. Nonetheless, throughout successive fractionations of neglect, reported cases of bidirectional task-specific neglect after unilateral brain damage are rare. We now report two such cases. After right hemisphere stroke, the first patient reliably showed severe left neglect on cancellation but right neglect on line bisection. After left hemisphere stroke, the second case showed right neglect on cancellation but left neglect on line bisection. Extensive investigation of case 1 confirmed our previous conjecture that the crucial distinction between these tasks lies in the presence or the absence of an overt target. In contrast to cancellation, line bisection demands the internal computation of the location of the "target" (the midpoint), followed by executing a motor response toward the precise location of that "imaginary" midpoint. The relative attentional and premotor contributions of the intact and damaged hemispheres to these forms of bidirectional neglect are also assessed.     

Relationship between donor age and the replicative lifespan of human cells in culture: a reevaluation

Normal human diploid fibroblasts have a finite replicative lifespan in vitro, which has been postulated to be a cellular manifestation of aging in vivo. Several studies have shown an inverse relationship between donor age and fibroblast culture replicative lifespan; however, in all cases, the correlation was weak, and, with few exceptions, the health status of the donors was unknown. We have determined the replicative lifespans of 124 skin fibroblast cell lines established from donors of different ages as part of the Baltimore Longitudinal Study of Aging. All of the donors were medically examined and were declared "healthy," according to Baltimore Longitudinal Study of Aging protocols, at the time the biopsies were taken. Both long- and short-lived cell lines were observed in all age groups, but no significant correlation between the proliferative potential of the cell lines and donor age was found. A comparison of multiple cell lines established from the same donors at different ages also failed to reveal any significant trends between proliferative potential and donor age. The rate of [3H]thymidine incorporation and the initial rates of growth during the first few subcultivations were examined in a subset of cell lines and were found to be significantly greater in fetal lines than in postnatal lines. Cell lines established from adults did not vary significantly either in initial growth rate or in [3H]thymidine incorporation. These results clearly indicate that, if health status and biopsy conditions are controlled, the replicative lifespan of fibroblasts in culture does not correlate with donor age.     

Obturation of a C-shaped canal using an improved method of warm lateral condensation

Pilot studies conducted by the authors seem to indicate that obturation of a root canal system by the lateral condensation of gutta-percha using an electrically heated spreader results in a more homogeneous mass of gutta-percha than standard lateral condensation. This article describes an improved variation which prevents inadvertent dislodgement of the mass of gutta-percha during the obturation. The authors refer to this technique as "zap and tap" and a case report describes its use to obturate a C-shaped canal system of a mandibular second molar.     

Hypersensitivity interstitial pneumopathy and ulcero-hemorrhagic rectocolitis: role of methotrexate

The authors report the case of a 46-year-old man with refractory ulcerative colitis treated with methotrexate who was admitted in the hospital for asthenia, fever, cough and dyspnea. Owing to the development of adult respiratory distress syndrome despite broad spectrum antibiotherapy, the patient was transferred to the intensive care unit. A diagnosis of pneumonitis due to methotrexate was made. Patient's condition improved after discontinuation of the drug, mechanical ventilation, and corticosteroids. The increasing use of methotrexate in several gastroenterological diseases warrants further consideration of the potential devastating side effects of this drug, particularly on the lungs. A review of the literature on this topic is provided in the "discussion" section.     

Nonsanction seat belt law enforcement: a modern day tale of two cities

This paper describes the implementation and evaluation of a nonsanction seat belt law enforcement program in two experimental communities of contrasting size in a state with a mandatory belt law. The main ingredients of the program were seat belt "salutes," public information and education, and limited use of inexpensive economic incentives. Driver shoulder belt use data collected before, during, and after the experimental programs, compared to similar data collected in a comparison community, showed the approach to be effective. While standard seat belt enforcement activities without incentives have been shown to be effective, many police departments, especially in smaller communities, are reluctant to make wholesale increases in seat belt citations. Although requiring some additional level of manpower and resources, a nonsanction approach to seat belt law enforcement can provide an alternate way of increasing belt use in these communities.