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This work presents a one-step synthesis methodology for preparing a hydrochar (HC) doped with TiO 2 (HC-TiO 2 ) for its application on the degradation of crystal violet (CV) using UV and visible radiation. Byrsonima crassifolia stones were used as precursors along with TiO 2 particles. The HC-TiO 2 sample was synthesized at 210 °C for 9 h using autogenous pressure. The photocatalyst was characterized to evaluate the TiO 2 dispersion, specific surface area, graphitization degree, and band-gap value. Finally, the degradation of CV was investigated by varying the operating conditions of the system, the reuse of the catalyst, and the degradation mechanism. The physicochemical characterization of the HC-TiO 2 composite showed good dispersion of TiO 2 in the carbonaceous particle. The presence of TiO 2 on the hydrochar surface yields a bandgap value of 1.17 eV, enhancing photocatalyst activation with visible radiation. The degradation results evidenced a synergistic effect with both types of radiation due to the hybridized π electrons in the sp 2 -hybridized structures in the HC surface. The degradation percentages were on average 20% higher using UV radiation than visible radiation under the following conditions: [CV] = 20 mg/L, 1 g/L of photocatalyst load, and pH = 7.0. The reusability experiments demonstrated the feasibility of reusing the HC-TiO 2 material up to 5 times with a similar photodegradation percentage. Finally, the results indicated that the HC-TiO 2 composite could be considered an efficient material for the photocatalytic treatment of water contaminated with CV., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jonathan M. Sanchez-Silva reports financial support was provided by National Council on Science and Technology., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Introduction: Laryngopharyngeal reflux (LPR) is difficult to diagnose and treat owing to uncertainty relating to the underlying pathology. The initial management of LPR includes lifestyle modifications and oral medications. In patients who have failed to respond to proton pump inhibitor (PPI) therapy, anti-reflux surgery is considered; laparoscopic fundoplication is the surgery of choice. The primary aim of this review is to identify whether fundoplication is effective in improving signs and symptoms of LPR. The secondary aim is to identify whether patients who have had a poor response to PPIs are likely to have symptom improvement with surgery. The objective of the study is to establish the effect of laparoscopic fundoplication on the reflux symptom index score (RSI)., Methods: PubMed, Embase, Medline and Cochrane databases were used to search according to the PRISMA guidelines. Original articles assessing the efficacy of fundoplication in relieving symptoms of LPR were included. For each study, the efficacy endpoints and safety outcomes were recorded., Findings: Nine studies from 844 initial records met the inclusion criteria: one prospective case control study, one retrospective case-control study, four prospective case series and three retrospective case series involving 287 fundoplications. All nine studies found fundoplication to be effective in improving symptoms of LPR ( p  < 0.05)., Conclusion: Current evidence suggests laparoscopic fundoplication is an effective treatment for LPR and should be considered if medical management is unsuccessful.

Background: The Duke Activity Status Index (DASI) questionnaire might help incorporate self-reported functional capacity into preoperative risk assessment. Nonetheless, prognostically important thresholds in DASI scores remain unclear. We conducted a nested cohort analysis of the Measurement of Exercise Tolerance before Surgery (METS) study to characterise the association of preoperative DASI scores with postoperative death or complications., Methods: The analysis included 1546 participants (≥40 yr of age) at an elevated cardiac risk who had inpatient noncardiac surgery. The primary outcome was 30-day death or myocardial injury. The secondary outcomes were 30-day death or myocardial infarction, in-hospital moderate-to-severe complications, and 1 yr death or new disability. Multivariable logistic regression modelling was used to characterise the adjusted association of preoperative DASI scores with outcomes., Results: The DASI score had non-linear associations with outcomes. Self-reported functional capacity better than a DASI score of 34 was associated with reduced odds of 30-day death or myocardial injury (odds ratio: 0.97 per 1 point increase above 34; 95% confidence interval [CI]: 0.96-0.99) and 1 yr death or new disability (odds ratio: 0.96 per 1 point increase above 34; 95% CI: 0.92-0.99). Self-reported functional capacity worse than a DASI score of 34 was associated with increased odds of 30-day death or myocardial infarction (odds ratio: 1.05 per 1 point decrease below 34; 95% CI: 1.00-1.09), and moderate-to-severe complications (odds ratio: 1.03 per 1 point decrease below 34; 95% CI: 1.01-1.05)., Conclusions: A DASI score of 34 represents a threshold for identifying patients at risk for myocardial injury, myocardial infarction, moderate-to-severe complications, and new disability., (Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The main purpose of this study was to explore the potential influences of pickleweed vegetation on the abundance, diversity and metabolic activities of microbial communities in four distinct areas of a petroleum-contaminated solid waste management unit (SWMU) located in Contra Costa County, northern California. The four areas sampled include two central areas, one of which is central vegetated (CV) and one unvegetated (UV), and two peripheral vegetated areas, one of which is located to the west side of the SWMU (V-West) and one located to the east side (V-East). Measurements were made of total petroleum hydrocarbons (TPH), polyaromatic hydrocarbons (PAH), soil physicochemical properties, and various aspects of microbial communities including metabolic activities, microbial abundances (PLFAs), diversity and composition based on amplicon sequencing. The peripheral V-East and V-West sites had 10-times lower electrical conductivity (EC) than that of the CV and UV sites. The high salinity levels of the CV and UV sites were associated with significant reductions in bacterial and fungal abundances (PLFA) when compared to V-East but not when compared to V-West. TPH levels of CV and UV were not significantly different from those of V-West but were substantially lower than V-East TPH (19,311 mg/kg of dry soil), the high value of which may have been associated with a pipeline that ran through the area. Microbial activities (in terms of soil respiration and the activities of three soil enzymes, i.e., urease, lipase, and phosphatase) were greatest in the vegetated sites compared to the UV site. The prokaryotic community was not diverse as revealed by the Shannon index with no significant variation among the four groups of samples. However, the fungal community of the peripheral sites, V-East and V-West had significantly higher OTU richness and Shannon index. Structure of prokaryotic communities inhabiting the rhizosphere of pickleweed plants at the three sites differed significantly and were also different from those found in the UV region of the central site according to pairwise, global PERMANOVA and ANOSIM analyses. The differences in OTU-based rhizosphere-associated bacterial and fungal communities' composition were explained mainly by the changes in soil EC and pH. The results suggest that saline TPH-contaminated areas that are vegetated with pickleweed are likely to have increased abundances, diversity and metabolic activities in the rhizosphere compared to unvegetated areas, even in the presence of high salinity., Competing Interests: Author BLP is an employee of the commercial company PG&E. The authors declare that no competing interests exist. The affiliation with PG&E does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

Background and Purpose: While there have been recent reports of brain retention of gadolinium following gadolinium-based contrast agent administration in adults, a retrospective series of pediatric patients has not previously been reported, to our knowledge. We investigated the relationship between the number of prior gadolinium-based contrast agent doses and increasing T1 signal in the dentate nucleus on unenhanced T1-weighted MR imaging. We hypothesized that despite differences in pediatric physiology and the smaller gadolinium-based contrast agent doses that pediatric patients are typically administered based on weighted-adjusted dosing, the pediatric brain would also demonstrate dose-dependent increasing T1 signal in the dentate nucleus., Materials and Methods: We included children with multiple gadolinium-based contrast agent administrations at our institution. A blinded reader placed ROIs within the dentate nucleus and adjacent cerebellar white matter. To eliminate reader bias, we also performed automated ROI delineation of the dentate nucleus, cerebellar white matter, and pons. Dentate-to-cerebellar white matter and dentate-to pons ratios were compared with the number of gadolinium-based contrast agent administrations., Results: During 20 years at our institution, 280 patients received at least 5 gadolinium-based contrast agent doses, with 1 patient receiving 38 doses. Sixteen patients met the inclusion/exclusion criteria for ROI analysis. Blinded reader dentate-to-cerebellar white matter ratios were significantly associated with gadolinium-based contrast agent doses (r s = 0.77, P = .001). The dentate-to-pons ratio and dentate-to-cerebellar white matter ratios based on automated ROI placement were also significantly correlated with gadolinium-based contrast agent doses (t = 4.98, P < .0001 and t = 2.73, P < .02, respectively)., Conclusions: In pediatric patients, the number of prior gadolinium-based contrast agent doses is significantly correlated with progressive T1-weighted dentate hyperintensity. Definitive confirmation of gadolinium deposition requires tissue analysis. Any potential clinical sequelae of gadolinium retention in the developing brain are unknown. Given this uncertainty, we suggest taking a cautious stance, including the use, in pediatric patients, of higher stability, macrocyclic agents, which in both human and animal studies have been shown to be associated with lower levels of gadolinium deposition, and detailed documentation of dosing. Most important, a patient should not be deprived of a well-indicated contrasted MR examination., (© 2016 by American Journal of Neuroradiology.)

Introduction: This study aimed to examine the relationship between gender, apolipoprotein E (APOE) genotype, and mesial temporal atrophy in mild cognitive impairment (MCI) with and without progression to Alzheimer's disease (AD)., Methods: We evaluated 236 MCI patients with (n = 121) and without (n = 115) AD progression. Longitudinal MRI-based hippocampal volumes (HV) and entorhinal cortex (ERC) thickness were obtained. The Clinical Dementia Rating Sum of Boxes (CDR-SB) score was used to assess disease severity., Results: We found a significant effect of APOE, gender, and clinical course (stable MCI versus MCI-AD progression) on HV. There was a significant effect of clinical course and APOE, but not gender, on ERC. Baseline HV and APOE4 status predicted MCI-AD progression in women. Baseline ERC and APOE4 status predicted MCI-AD progression in men. There were significant differences in CDR-SB scores between patients with and without MCI-AD progression, but not between males and females, or APOE4 carriers and non-carriers., Conclusions: HV, but not ERC, is strongly influenced by gender in MCI. The effects of gender and APOE4 on neuroimaging biomarkers have potentially important implications in the prediction of MCI-AD progression and should be taken into account in clinical trials.

Aim: To document the radiation exposure metrics, including fluoroscopic radiation time and radiation dose-area product, in children <18 years of age who undergo nasoenteral tube placement using fluoroscopic guidance with maximal dose-reduction techniques., Materials and Methods: Following institutional review board approval, the age, gender, anatomical information, immediate procedure-related complications if any, fluoroscopy time, and radiation dose-area product were collected retrospectively in all paediatric patients who underwent fluoroscopically guided nasoenteral tube placement during a 5-year period. Three paediatric radiology faculty members, a radiologist assistant, and trainee residents during their paediatric radiology rotation performed the procedures on two different digital fluoroscopic machines using radiation-minimising techniques. Median values of the fluoroscopy time and radiation dose-area product were calculated and compared to values reported in the literature using the Wilcoxon procedure., Results: There were 41 male and 33 female patients with a median age of 4 years and 6 months. Median fluoroscopy time used for placing a nasoenteral tube was 1.25 minutes with a median radiation exposure dose-area product of 0.245 Gy·cm(2). All patients had successful placement of nasoenteral tube without immediate procedure-related complications., Conclusion: Fluoroscopy-guided nasoenteral feeding tube placement can be performed successfully with minimal radiation exposure without compromising procedural success., (Copyright © 2016 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved.)

The development of clinically acceptable albumin-based nanoparticle formulations for use in pulmonary drug delivery has been hindered by concerns about the toxicity of nanomaterials in the lungs combined with a lack of information on albumin nanoparticle clearance kinetics and biodistribution. In this study, the in vivo biocompatibility of albumin nanoparticles was investigated following a single administration of 2, 20, and 390μg/mouse, showing no inflammatory response (TNF-α and IL-6, cellular infiltration and protein concentration) compared to vehicle controls at the two lower doses, but elevated mononucleocytes and a mild inflammatory effect at the highest dose tested. The biodistribution and clearance of (111)In labelled albumin solution and nanoparticles over 48h following a single pulmonary administration to mice was investigated by single photon emission computed tomography and X-ray computed tomography imaging and terminal biodistribution studies. (111)In labelled albumin nanoparticles were cleared more slowly from the mouse lung than (111)In albumin solution (64.1±8.5% vs 40.6±3.3% at t=48h, respectively), with significantly higher (P<0.001) levels of albumin nanoparticle-associated radioactivity located within the lung tissue (23.3±4.7%) compared to the lung fluid (16.1±4.4%). Low amounts of (111)In activity were detected in the liver, kidneys, and intestine at time points >24h indicating that small amounts of activity were cleared from the lungs both by translocation across the lung mucosal barrier, as well as mucociliary clearance. This study provides important information on the fate of albumin vehicles in the lungs, which may be used to direct future formulation design of inhaled nanomedicines., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Activator protein-1 (AP-1) is a ubiquitous transcription factor that paradoxically also has some tissue-specific functions. In skeletal muscle cells, we document that the AP-1 subunit, Fra-2, is expressed in the resident stem cells (Pax7-positive satellite cells) and also in the analogous undifferentiated 'reserve' cell population in myogenic cultures, but not in differentiated myofiber nuclei. Silencing of Fra-2 expression enhances the expression of differentiation markers such as muscle creatine kinase and myosin heavy chain, indicating a possible role of Fra-2 in undifferentiated myogenic progenitor cells. We observed that Fra-2 is a target of cytokine-mediated extracellular signal-regulated kinase-1/2 signaling in cultured muscle cells, and extensive mass spectrometry and mutational analysis identified S320 and T322 as regulators of Fra-2 protein stability. Interestingly, Fra-2 S320 phosphorylation occurs transiently in activated satellite cells and is extinguished in myogenin-positive differentiating cells. Thus, cytokine-mediated Fra-2 expression and stabilization is linked to regulation of myogenic progenitor cells having implications for the molecular regulation of adult muscle stem cells and skeletal muscle regeneration.

Satellite cells, originating in the embryonic dermamyotome, reside beneath the myofibre of mature adult skeletal muscle and constitute the tissue-specific stem cell population. Recent advances following the identification of markers for these cells (including Pax7, Myf5, c-Met and CD34) (CD, cluster of differentiation; c-Met, mesenchymal epithelial transition factor) have led to a greater understanding of the role played by satellite cells in the regeneration of new skeletal muscle during growth and following injury. In response to muscle damage, satellite cells harbour the ability both to form myogenic precursors and to self-renew to repopulate the stem cell niche following myofibre damage. More recently, other stem cell populations including bone marrow stem cells, skeletal muscle side population cells and mesoangioblasts have also been shown to have myogenic potential in culture, and to be able to form skeletal muscle myofibres in vivo and engraft into the satellite cell niche. These cell types, along with satellite cells, have shown potential when used as a therapy for skeletal muscle wasting disorders where the intrinsic stem cell population is genetically unable to repair non-functioning muscle tissue. Accurate understanding of the mechanisms controlling satellite cell lineage progression and self-renewal as well as the recruitment of other stem cell types towards the myogenic lineage is crucial if we are to exploit the power of these cells in combating myopathic conditions. Here we highlight the origin, molecular regulation and therapeutic potential of all the major cell types capable of undergoing myogenic differentiation and discuss their potential therapeutic application.

The case report is presented of a patient with West Nile virus infection and ventilator dependent respiratory failure in whom bilateral diaphragmatic paralysis developed. If the prevalence of West Nile virus infection continues to rise, recognition of diaphragmatic paralysis and related respiratory complications will become increasingly important to the pulmonary/critical care physician.

Objectives: To investigate relationships between declining owned-cat populations and cat-related clinical work and practice activities., Design: Sydney practitioners were interviewed, and practice records were examined, from small animal veterinary practices (n = 56) in Sydney. Using data that covered a 4 year period, the age distribution of cats was examined and associated trends in cat-related clinical work were analysed., Results: Important changes in the pet population attending veterinary practices were found, with 38.2% of respondents reporting a fall in cat desexings from 1996 to 2000., Conclusions: While economic impact could not be determined, the qualitative data demonstrated that the majority of practices in the survey were active in the promotion of cat ownership. The study provides a useful model for future investigations. To facilitate this process and to enhance principals' knowledge of their practice's population dynamics, we advocate a minimum standard of data collection that records week-by-week activities and the deaths of pets.

Matrilysin, a member of matrix metalloproteinase family, is believed to play a significant role in the growth and proliferation of colon cancer cells. Overexpression of the matrilysin gene has been shown to correlate with Dukes' stage and increased metastatic potential in colorectal cancer. The aim of this study was to evaluate the effect of preoperative high-dose radiotherapy (25 Gy in five fractions over 5 days) on matrilysin (MMP-7) gene expression, in patients with resectable rectal cancer, by a quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Biopsy samples of tumour (n=30) and distant normal mucosa (n=12) from 15 patients were obtained pre- and post-radiotherapy. Messenger (m)RNA was extracted from all of the tissue samples and reverse transcribed to double-stranded cDNA. Quantitative RT-PCR was performed to study the effect of preoperative radiotherapy on matrilysin gene expression in both the tumour and normal mucosal specimens. Matrilysin mRNA values were expressed relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for each sample. In 14 out of 15 cases, matrilysin mRNA was detected in the cancerous tissue. Although all six normal mucosal specimens expressed matrilysin mRNA, the levels were approximately 10-fold lower compared with those seen in the paired tumour samples. Preoperative radiotherapy led to a significant 6- to 7-fold increase (P=0.001) in the expression of matrilysin mRNA in rectal cancer tissue. In contrast, there was no significant change in the matrilysin mRNA expression of normal mucosal specimens post-radiotherapy. Preoperative high-dose radiotherapy upregulates matrilysin gene expression in rectal cancer. Matrilysin inhibition may be a useful preventive or therapeutic adjunct to radiotherapy in rectal cancer.

Metastasis is the final step in tumor progression from a benign cell to a fully malignant cell. The metastatic phenotype results from a wide range of phenotypic changes in the cell from the expression of proteinases, to adhesion molecules, the loss of proteinase inhibitors and tumor suppressor gene function, to name a few. However, the molecular basis for this progression has long been investigated and there does not appear to be a specific genetic alteration responsible for influencing all the changes which occur in a metastatic cell. As mentioned, the proteolytic ability of the cell is a key factor in the malignant phenotype and the expression of matrix metalloproteinases (MMPs) is known to contribute to metastases (1). The gelatinase group (MMP-2 and MMP-9) within this enzyme family has been associated with tumor progression and the active form of MMP-2 has the strongest correlation with the metastatic phenotype in colorectal cancer (2).

Objective: To evaluate the long-term efficacy and tolerability within primary care settings of orlistat, a gastrointestinal lipase inhibitor, for the treatment of obesity., Design: Randomized, double-blind, placebo-controlled, multicenter study., Participants: A group of 796 obese patients (body mass index, 30-44 kg/m2), treated with placebo 3 times a day (TID), 60 mg of orlistat TID, or 120 mg of orlistat TID, in conjunction with a reduced-energy diet for the first year and a weight-maintenance diet during the second year., Setting: Seventeen primary care centers in the United States., Main Outcome Measures: Changes in body weight and obesity-related disease risk factors., Results: Patients treated with orlistat lost significantly more weight (7.08 +/- 0.54 and 7.94 +/- 0.57 kg for the 60-mg and 120-mg orlistat groups, respectively) than those treated with placebo (4.14 +/- 0.56 kg) in year 1 (P<.001) and sustained more of this weight loss during year 2 (P<.001). More patients treated with orlistat lost 5% or more of their initial weight in year 1 (48.8% and 50.5% of patients in the 60-mg and 120-mg groups, respectively) compared with placebo (30.7%; P<.001), and approximately 34% of patients in the orlistat groups sustained weight loss of 5% or greater over 2 years compared with 24% in the placebo group (P<.001). Orlistat produced greater improvements than placebo in serum lipid levels and blood pressure and was well tolerated, although treatment resulted in a higher incidence of gastrointestinal events., Conclusions: This long-term study indicates that orlistat is an effective adjunct to dietary intervention in the treatment of obesity in primary care settings.

Donor lymphocyte infusions (DLI) can induce remissions in patients who have relapsed after allogeneic bone marrow transplantation (BMT). However, DLI frequently also result in significant acute and/or chronic graft-versus-host disease (GVHD). Several clinical and experimental lines of evidence have suggested that CD8(+) T cells play a critical role in the pathogenesis of GVHD. To develop methods to reduce the incidence of GVHD associated with DLI, we administered defined numbers of CD4(+) donor T cells after ex vivo depletion of CD8(+) lymphocytes to 40 patients with relapsed hematologic malignancies after allogeneic BMT. Cohorts of patients received 0.3, 1.0, or 1.5 x 10(8) CD4(+) cells/kg. Overall, 12 of 38 patients (32%) evaluable for toxicity developed acute or chronic GVHD. However, 6 of 27 patients (22%) receiving 0.3 x 10(8) CD4 cells/kg developed GVHD compared with 6 of 11 patients (55%) who received >/=1.0 x 10(8) CD4 cells/kg (P = .07). Treatment-related mortality was low (3%), with 1 death related to infection in the setting of immunosuppression for GVHD. Disease responses after CD4(+) DLI were documented in 15 of 19 patients (79%) with early-phase chronic myelogenous leukemia (CML) relapse, 5 of 6 patients (83%) with relapsed multiple myeloma, and 1 patient with myelodysplasia. For patients with early-phase CML relapse, the Kaplan-Meier probability of achieving complete cytogenetic remission was 87% and the probability of complete molecular response was 78% at 1 year after DLI. The median time to complete cytogenetic response and molecular response in patients with CML was 13 weeks (range, 9 to 30 weeks) and 34 weeks (range, 10 to 56 weeks), respectively. The median time to response in patients with multiple myeloma was 26 weeks (range, 15 to 62 weeks). All patients in this trial who developed GVHD demonstrated tumor regression, but the presence of GVHD was not required for patients to achieve a response, because 48% of responding patients never developed evidence of GVHD. Two patients with CML who did not respond at dose level 1 subsequently achieved complete cytogenetic remission after a second infusion of CD8-depleted cells at dose level 2. In patients with evidence of mixed hematopoietic chimerism who achieved a complete remission after DLI, cytogenetic analysis of marrow cells also demonstrated conversion to complete donor hematopoiesis in all evaluable patients. These studies suggest that relatively low numbers of CD8-depleted donor lymphocytes are effective in inducing complete remissions in patients with stable-phase CML and multiple myeloma who have relapsed after allogeneic BMT. Because of the relatively low risk of toxicity associated with the infusion of defined numbers of CD4(+) donor cells, further studies can be undertaken in the setting of persistent minimal residual disease to prevent relapse after allogeneic BMT.

A hospital-driven IDS that encounters serious problems resulting from ownership of a physician practice should address those problems by focusing on three core areas: vision and leadership, effectiveness of operations, and physician compensation arrangements. If changes in these areas do not lead to improvements, the IDS may need to consider organizational restructuring. In one case study, a hospital-driven IDS faced the problem of owning a poorly performing MSO with a captive physician group. The IDS's governing board determined that the organization lacked effective communication with the physicians and that realization of the organization's vision would require greater physician involvement in organizational decision making. The organization is expected to undergo some corporate reorganization in which physicians will acquire an equity interest in the enterprise.

The success of mycobacteria as pathogens hinges on their ability to infect and persist within the macrophages of their host. However, activation of host macrophages by cytokines from a productive cellular immune response can stimulate the cells to kill their resident pathogens. This suggests that the interaction between host cell and microbe is in delicate balance, which can be tipped in favour of either organism. Biochemical analysis of mycobacterial vacuoles has shown them to be integral to the host cell's recycling endosomal system. As such they show limited acidification and hydrolytic activity despite possession of known lysosomal constituents such as cathepsins D, B and L, and LAMP 1. Even in established infections, they remain dynamic compartments accessible to several plasmalemma-derived constituents. Once the macrophage has been activated by IFN-gamma and TNF-alpha the vacuoles coalesce and acidify. This marks a distinct alteration in vacuole physiology and leads to stasis and death of the mycobacteria. Mycobacteria have developed several strategies to avoid this outcome. Most notably, live bacilli-induce sustained release of IL-6 from infected macrophages. IL-6 blocks the ability of both polyclonal primary T cells and T-cell hybridomas to respond to appropriate stimuli. Such an activity could render the centres of infection foci, such as granulomas, anergic and thus avoid release of macrophage-activating cytokines. This paper discusses both the mechanisms by which mycobacteria try to ensure their success as intracellular pathogens and the relevance of these strategies to the overall understanding of mycobacterial diseases.

The clinical efficacy of donor lymphocyte infusions (DLI) in patients with relapsed chronic myelocytic leukemia after allogeneic bone marrow transplantation has been demonstrated in several recent studies. Although it is presumed that allogeneic T cells mediate this graft-versus-leukemia (GVL) effect, the influence of DLI on the T cell compartment of recipients has not been determined. To characterize the immunologic effects of DLI and to identify T cell changes selectively associated with the GVL response, we analyzed the T cell receptor (TCR) repertoire in four patients with relapsed chronic myelocytic leukemia who achieved a complete remission after infusion of CD4+ lymphocytes from HLA-identical sibling donors. Only one of the four patients developed clinically significant graft-versus-host disease (GVHD) after infusion of donor lymphocytes. TCR repertoire was examined after PCR amplification of 24 Vbeta gene subfamilies in serial samples obtained over a 1-yr period before and after DLI. Results were compared to 10 normal donors. Before DLI, all four patients were found to have abnormal TCR Vbeta repertoire in peripheral T cells, associated with a large number of clonal and oligoclonal patterns. Abnormal TCR patterns persisted for at least 3 mo after DLI, but thereafter gradually began to normalize. By 1 yr after DLI, all patients demonstrated almost complete normalization of Vbeta repertoire with polyclonal representation within almost all Vbeta gene subfamilies. We also examined changes in the TCR Vbeta repertoire associated with the disappearance of Ph+ cells. In each patient, we were able to identify the expansion of at least 1 Vbeta gene subfamily that coincided with the time of the cytogenetic response. In one patient who was studied in greater detail, CDR3 size analysis of serial samples after DLI indicated that these changes were associated with the appearance of clonal T cells. This finding was confirmed through CDR3 sequence analysis and use of CDR3 clone-specific oligonucleotide probes. A putative GVL clone identified by this technique was not detectable in either donor or patient T cells before DLI, but persisted in peripheral T cells for approximately 1 yr. These experiments therefore provide evidence for the clonal expansion of allogeneic T cells that may be selective mediators of antileukemia activity without also mediating graft-versus-host disease.

R24 is a monoclonal antibody that recognizes the disialoganglioside GD3 expressed on the surface of malignant melanoma cells. Once bound, it can mediate destruction of these cells through both complement-mediated lysis and antibody-dependent cellular cytotoxicity. Agents such as interleukin 2 (IL-2), which can augment effector cell function and promote destruction of antibody-coated tumor cells, might produce improved antitumor responses when combined with R24. In this series, we evaluated the combination of R24 and IL-2 in a Phase 1b study in patients with metastatic melanoma. Twenty-eight patients with metastatic melanoma were entered into the protocol at two institutions. Patients received 8 weeks of IL-2 by continuous i.v. infusion at a dose (4.5 x 10(5) Amgen units/m2/day) designed to selectively expand natural killer (NK) cells. In weeks 5 and 6, patients received R24 for a total of four doses. Twenty-four h after each R24 infusion, patients received a 2-h bolus dose of IL-2 to help promote activity of NK effectors against antibody-coated melanoma targets. Additional IL-2 boluses were administered in weeks 7 and 8. Doses were escalated through two bolus doses of R24 (5 or 15 mg/m2) and two bolus doses of IL-2 (2.5 or 5.0 x 10(5) units/m2). Although one patient experienced severe capillary leak syndrome during IL-2, therapy was otherwise well tolerated. At the higher dose level of R24, two of four patients experienced transient but severe abdominal and chest discomfort, necessitating dose reduction. One patient with ocular melanoma and liver metastases had a partial response. Two additional patients had minor responses. A dramatic increase in NK cell number was noted as a result of treatment, as was augmentation of cytolytic activity against cultured NK-sensitive targets. Antibody-dependent cellular cytotoxicity against cultured melanoma cells in the presence of exogenous R24 or in the presence of serum obtained from patients following R24 infusion also increased during treatment. Our experience indicates that R24 and low-dose IL-2 can be safely combined in patients with metastatic melanoma and that this combination can promote destruction of cultured melanoma cells. The clinical activity of this combination against ocular melanoma may merit further investigation.