Improved LINE-1 Detection through Pattern Matching by Increasing Probe Length.

Long Interspersed Element-1 (LINE-1 or L1) is an autonomous transposable element that accounts for 17% of the human genome. Strong correlations between abnormal L1 expression and diseases, particularly cancer, have been documented by numerous studies. L1PD (LINE-1 Pattern Detection) had been previously created to detect L1s by using a fixed pre-determined set of 50-mer probes and a pattern-matching algorithm. L1PD uses a novel seed-and-pattern-match strategy as opposed to the well-known seed-and-extend strategy employed by other tools. This study discusses an improved version of L1PD that shows how increasing the size of the k-mer probes from 50 to 75 or to 100 yields better results, as evidenced by experiments showing higher precision and recall when compared to the 50-mers. The probe-generation process was updated and the corresponding software is now shared so that users may generate probes for other reference genomes (with certain limitations). Additionally, L1PD was applied to other non-human genomes, such as dogs, horses, and cows, to further validate the pattern-matching strategy. The improved version of L1PD proves to be an efficient and promising approach for L1 detection.

Complex alterations in inflammatory pain and analgesic sensitivity in young and ageing female rats: involvement of ASIC3 and Nav1.8 in primary sensory neurons.

OBJECTIVE AND DESIGN: Our aim was to determine an age-dependent role of Nav1.8 and ASIC3 in dorsal root ganglion (DRG) neurons in a rat pre-clinical model of long-term inflammatory pain. METHODS: We compared 6 and 24 months-old female Wistar rats after cutaneous inflammation. We used behavioral pain assessments over time, qPCR, quantitative immunohistochemistry, selective pharmacological manipulation, ELISA and in vitro treatment with cytokines. RESULTS: Older rats exhibited delayed recovery from mechanical allodynia and earlier onset of spontaneous pain than younger rats after inflammation. Moreover, the expression patterns of Nav1.8 and ASIC3 were time and age-dependent and ASIC3 levels remained elevated only in aged rats. In vivo, selective blockade of Nav1.8 with A803467 or of ASIC3 with APETx2 alleviated mechanical and cold allodynia and also spontaneous pain in both age groups with slightly different potency. Furthermore, in vitro IL-1ß up-regulated Nav1.8 expression in DRG neurons cultured from young but not old rats. We also found that while TNF-α up-regulated ASIC3 expression in both age groups, IL-6 and IL-1ß had this effect only on young and aged neurons, respectively. CONCLUSION: Inflammation-associated mechanical allodynia and spontaneous pain in the elderly can be more effectively treated by inhibiting ASIC3 than Nav1.8.

Gelatin methacryloyl hydrogel with and without dental pulp stem cells for TMJ regeneration: An in vivo study in rabbits.

BACKGROUND: In the last decade, tissue-engineering strategies for regenerating the temporomandibular joint (TMJ) have been investigated. This may be a promising strategy for the minimally invasive restoration of joint integrity. OBJECTIVES: To evaluate whether dental pulp stem cells (DPSCs) loaded in a light-occured hydrogel made of gelatin methacryloyl (GelMA) enhance the regeneration of osteochondral defects in the rabbit TMJ. MATERIALS AND METHODS: Defects were filled with GelMA alone (control group; n = 4) or filled with GelMA loaded with rabbit DPSCs (experimental group; n = 4), In one group, the TMJ capsule was opened without creating a defect (sham group; n = 2). The following micro-CT parameters were analysed: bone volume to total volume ratio (BV/TV%) and bone mineral density (BMD). Histological evaluation was performed to assess cartilage regeneration features. A semi-quantitative scoring system was also used to evaluate the defects. RESULTS: All groups had no statistical difference regarding the micro-CT parameters. The highest mean healing score was found for the experimental group. After 4 weeks, there were no signs of hydrogel in either group or no signs of inflammation in the adjacent tissues. The tissue formed in the defect was dense fibrous connective tissue. CONCLUSION: Adding DPSCs to GelMA did not provide a regenerative enhancement in TMJ osteochondral defects. This resulted in similar micro-CT parameters after 4 weeks of healing, with improved signs of subchondral bone regeneration but no cartilage regeneration.

Age-dependent and modality-specific changes in the phenotypic markers Nav1.8, ASIC3, P2X3 and TRPM8 in male rat primary sensory neurons during healthy aging.

The effects during healthy aging of the tetrodotoxin-resistant voltage-gated sodium channel 1.8 (Nav1.8), the acid-sensing ion channel-3 (ASIC3), the purinergic-receptor 2X3 (P2X3) and transient receptor potential of melastatin-8 (TRPM8) on responses to non-noxious stimuli are poorly understood. These effects will influence the transferability to geriatric subjects of findings obtained using young animals. To evaluate the involvement of these functional markers in mechanical and cold sensitivity to non-noxious stimuli and their underlying mechanisms, we used a combination of immunohistochemistry and quantitation of immunostaining in sub-populations of neurons of the dorsal root ganglia (DRG), behavioral tests, pharmacological interventions and Western-blot in healthy male Wistar rats from 3 to 24 months of age. We found significantly decreased sensitivity to mechanical and cold stimuli in geriatric rats. These behavioural alterations occurred simultaneously with differing changes in the expression of Nav1.8, ASIC3, P2X3 and TRPM8 in the DRG at different ages. Using pharmacological blockade in vivo we demonstrated the involvement of ASIC3 and P2X3 in normal mechanosensation and of Nav1.8 and ASIC3 in cold sensitivity. Geriatric rats also exhibited reductions in the number of A-like large neurons and in the proportion of peptidergic to non-peptidergic neurons. The changes in normal sensory physiology in geriatric rats we report here strongly support the inclusion of aged rodents as an important group in the design of pre-clinical studies evaluating pain treatments.

Recurrent ST-Elevation Myocardial Infarction (STEMI) in Coronary Artery Aneurysm Secondary to Atherosclerosis.

We describe a rare case of coronary artery aneurysm (CAA) with recurrent ST-elevation myocardial infarction (STEMI) despite being on standard dual antiplatelet therapy (DAPT). A 47-year-old male presented with chest pain and was found to have inferior wall STEMI along with diffuse right coronary artery (RCA) ectasia and proximal RCA aneurysm, thrombotic occlusion, and dissection. He was managed with extensive thrombectomy, angioplasty, prolonged Heparinization, and DAPT. The patient went on to have a similar presentation nine months later with a recurrent inferior wall STEMI with proximal RCA aneurysm and thrombotic occlusion managed with thrombectomy and bare metal stent placement. He was placed on long-term anticoagulation and DAPT with no further recurrence of MI reported on follow-up.

Reservoir sedimentation and spatiotemporal land use changes in their watersheds: the case of two sub-catchments of the White Volta Basin.

Reservoir storage is compromised by sedimentation for which reason it has become an important matter in reservoir operation and management. While many studies have investigated sediment deposition rate in reservoirs, few have analyzed reservoir sedimentation from their catchment's land use change perspective. Based on bathymetric survey conducted on two reservoirs in the White Volta Basin in 2020 and analysis of four Landsat satellite imagery (1986, 1996, 2006, and 2020) within their watersheds, this study assessed the land cover change within the watersheds to draw inferences on the rate of sedimentation of the reservoirs located downstream of their catchments. The results revealed rapid sedimentation in the small-sized reservoir (Vea), with an annual sedimentation rate of 0.304% and a nominal sedimentation rate of 0.17% for the mid-sized reservoir (Tono). Furthermore, the savannah forest within the Vea catchment declined drastically from 29.4% (1985) to 9.9% (2020) influenced by the rapid expansion of farmlands from 18.7% to 47.9% within the same period, respectively. On the other hand, the savannah forest within the Tono catchment declined from 34.7% (1985) to 21.6% (2020) due to farmland expansion from 19.2% to 39% within the same period, respectively. The higher sedimentation rate observed in the small-sized reservoir was observed to be worsened by extensive tree cover removal in its catchment. Therefore, land cover characteristics within a watershed have a significant bearing on the rate of sedimentation in the reservoirs located downstream of their catchment. Hence, adopting a multi-sectorial approach to dealing with land use management is necessary to sustain reservoirs' storage.

Glial-derived neurotrophic factor regulates the expression of TREK2 in rat primary sensory neurons leading to attenuation of axotomy-induced neuropathic pain.

TREK2 is a member of the 2-pore domain family of K+ channels (K2P) preferentially expressed by unmyelinated, slow-conducting and non-peptidergic isolectin B4-binding (IB4+) primary sensory neurons of the dorsal root ganglia (DRG). IB4+ neurons depend on the glial-derived neurotrophic factor (GDNF) family of ligands (GFL's) to maintain their phenotype. In our previous work, we demonstrated that 7 days after spinal nerve axotomy (SNA) of the L5 DRG, TREK2 moves away from the cell membrane resulting in a more depolarised resting membrane potential (Em). Given that axotomy deprives DRG neurons from peripherally-derived GFL's, we hypothesized that they might control the expression of TREK2. Using a combination of immunohistochemistry, immunocytochemistry, western blotting, in vivo pharmacological manipulation and behavioral tests we examined the ability of the GFL's (GDNF, neurturin and artemin) and their selective receptors (GFRα1, GFRα2 and GFRα3) to regulate the expression and function of TREK2 in the DRG. We found that TREK2 correlated strongly with the three receptors normally and ipsilaterally for all GFR's after SNA. GDNF, but not NGF, neurturin or artemin up-regulated the expression of TREK2 in cultured DRG neurons. In vivo continuous, subcutaneous administration of GDNF restored the subcellular distribution of TREK2 ipsilaterally and reversed mechanical and cold allodynia 7 days after SNA. This is the first demonstration that GDNF controls the expression of a K2P channel in nociceptors. As TREK2 controls the Em of C-nociceptors affecting their excitability, our finding has therapeutic potential in the treatment of chronic pain.

Evaluación de la estabilización de fracturas expuestas de pierna grados I y II de Gustillo en la etapa aguda / Analysis of the stabilization of Gustillo grade I and II open tibia fractures in the acute stage

Introducción: La fijación interna de las fracturas de pierna expuestas en la etapa aguda, es decir, dentro de las 24 h del trauma es un tema controvertido. El objetivo de este estudio fue evaluar las infecciones asociadas a la colocación de clavos endomedulares en la etapa aguda y a la colocación diferida, en la fijación de fracturas expuestas de pierna grados I y II de Gustilo. materiales y métodos: Se realizó un estudio de cohorte retrospectivo sobre el tratamiento en la etapa aguda de los pacientes que ingresaron en el hospital con fracturas expuestas de pierna entre 2015 y 2018. Se analizó la tasa de infecciones durante los primeros 6 meses después de la cirugía y se comparó la fijación en la etapa aguda con la fijación diferida. Resultados: La fijación interna con clavos endomedulares en la etapa aguda, en pacientes con fracturas expuestas de pierna no aumentó, sino que disminuyó la tasa de infecciones en el control posoperatorio. Conclusión: El estudio avala la colocación de clavos endomedulares en la etapa aguda, en pacientes con fractura de tibia expuestas. Nivel de Evidencia: II

Introduction: The internal fixation of leg fractures exposed in acute, that is, within 24 hours of trauma is quite controversial. The objective of this work is to assess infections associated with acute intramedullary nailing fixation ­versus deferred fixation­ of Gustilo type I and II open fractures. Patients and Methods: A retrospective cohort study was conducted of the acute treatment of patients with open leg fractures admitted to the hospital between 2015 and 2018. The infection rate was analyzed during the first 6 postoperative months after intramedullary nailing, and acute fixation patients were compared against deferred fixation patients. Results: Acute internal fixation with intramedullary nail in patients with open leg fractures does not increase, but decreases, the infection rate in the postoperative control. Conclusion: The study supports acute intramedullary nailing in patients with open tibial fractures. Level of Evidence: II

Unexpected solvent impact in the crystallinity of praziquantel/poly(vinylpyrrolidone) formulations. A solubility, DSC and solid-state NMR study.

The saturation solubility of PVP:PZQ physical mixtures (PMs) and solid dispersions (SDs) prepared from ethanol (E/E) or ethanol/water (E/W) by the solvent evaporation method at 1:1, 2:1 and 3:1 ratio (w/w) was determined. The presence of PVP improves the solubility of PZQ (0.31±0.01mg/mL). A maximum of 1.29±0.03mg/mL of PZQ in solution was achieved for the 3:1 SD (E/E). The amount of PZQ in solution depends on the amount of polymer and on the preparation method. Solid-state NMR (ssNMR) and DSC were used to understand this behavior. Results show that PMs are a mixture of crystalline PZQ with the polymer, while SDs show different degrees of drug amorphization depending on the solvent used. For E/W SDs, PZQ exists in amorphous and crystalline states, with no clear correlation between the amount of crystalline PZQ and the amount of PVP. For E/E SDs, formulations with a higher percentage of PZQ are amorphous with the components miscible in domains larger than 3nm ((1)H ssNMR relaxation measurements). Albeit its higher saturation solubility, the 3:1 E/E PVP:PZQ sample has a significant crystalline content, probably due to the water introduced by the polymer. High PVP content and small crystal size account for this result.

[Lethargic encephalitis. Report of one case]. / Enfermedad de Von Economo: Caso clínico.

Lethargic encephalitis (LE) is a Central Nervous System disorder following an upper respiratory tract infection, characterized by sleep disturbances, clinical symptoms corresponding to basal ganglia involvement and in some cases, neuropsychiatric sequelae. We report a 18-year-old mole with a history of sinusitis treated with azithromycin, two weeks before, presenting with fever, headache, confusion and myoclonus. Urine analysis was positive for cannabis. Cerebro spinal fluid analysis showed mononuclear pleiocytosis (109xmm³) and an increase in protein concentration of l.6 g/dl. Forty eight hours after admission, the patient required mechanical ventilation and subsequently a status epilepticus appeared. Ten days later, fever, rigidity and resting tremor appeared. A magnetic resonance imaging showed hyperintensities in FLALR sequence in the right insular cortex. The patient continued with extreme rigidity, catatonia and mutism. Considering the possibility ofa LE, methyl prednisolone 1 g/day was administered for five consecutive days followed by prednisone 40 mg l day, observing a dramatic improvement of rigidity and tremors.

Enfermedad de Von Economo: Caso clínico / Lethargic Encephalitis: Report of one case

Lethargic encephalitis (LE) is a Central Nervous System disorder following an upper respiratory tract infection, characterized by sleep disturbances, clinical symptoms corresponding to basal ganglia involvement and in some cases, neuropsychiatric sequelae. We report a 18-year-old mole with a history of sinusitis treated with azithromycin, two weeks before, presenting with fever, headache, confusion and myoclonus. Urine analysis was positive for cannabis. Cerebro spinal fluid analysis showed mononuclear pleiocytosis (109xmm³) and an increase in protein concentration ofl.6 g/dl. Forty eight hours after admission, the patient required mechanical ventilation and subsequently a status epilepticus appeared. Ten days later, fever, rigidity and resting tremor appeared. A magnetic resonance imaging showed hyperintensities in FLALR sequence in the right insular cortex. The patient continued with extreme rigidity, catatonia and mutism. Considering the possibility ofa LE, methyl prednisolone 1 g/day was administered for five consecutive days followed by prednisone 40 mgl day, observing a dramatic improvement of rigidity and tremors.

High level of Schistosoma mansoni infection in pre-school children in Sierra Leone highlights the need in targeting this age group for praziquantel treatment.

BACKGROUND: Schistosomiasis and soil-transmitted helminth (STH) infections are endemic in Sierra Leone. The consequences of these diseases to pre-school children are well understood. The national control program currently does not target this group of children for schistosomiasis, while mass drug administration (MDA) has been performed six monthly for STHs in children 12-59 months of age since 2006. METHODS: To assist the national decision on MDA strategy to control schistosomiasis and STH, three cross-sectional surveys were conducted in pre-school children in 2009-2011 as part of routine surveillance performed in different areas and in different phases of MDA, including known 'Hard to Reach' villages where consistently poor coverage results were seen in recent MDA. Thirty 4-5 year-old children were randomly selected per site and a stool sample from each child was examined by Kato-Katz thick smear. Pooled data were analyzed for schistosomiasis and separate sets of data were presented for STHs. In total 61 sites were surveyed and a total of 1803 children were examined. RESULTS: The overall prevalence and intensity of Schistosoma mansoni was 11.2% (95% CI 9.7-12.8) and 33.5 epg (95% CI 19.7-47.3). Relatively high level of infection was found in Kono (35.4% and 102.9 epg), Tonkolili (30.4% and 142.3 epg) and Koinadugu (20.8% and 47.0 epg). There were 8.1% of children 4-5 years old moderately or heavily infected with S. mansoni. Overall level of STH infections were generally low, with hookworm 8.4-22.8%, Ascaris lumbricoides 0.2-17.2%, and Trichuris trichiura 0.9-2.6% in three surveys. However, prevalence of hookworm and A. lumbricoides was relatively high in those hard-to-reach villages even two months after MDA. CONCLUSIONS: Relatively high levels of S. mansoni infections were found in children aged 4-5 years old in Sierra Leone, in line with geographical distribution of the disease observed in older children in the country. The results suggest that this group of children should not be neglected further in the schistosomiasis MDA and a global guideline is needed. Overall prevalence of STH infection was relatively low. Although there was no baseline data for direct comparison, it did show a marked reduction in STH infections, compared with historical data. However, relatively higher prevalence in hard-to-reach villages suggests the difficulty and quality of implementing MDA in such difficult locations, and more efforts and perhaps different delivery strategies are needed in these locations to increase the quality of MDA.

Magnetic resonance imaging: a wealth of cardiovascular information.

Cardiac magnetic resonance imaging is a relatively new noninvasive imaging modality that provides insight into multiple facets of the human myocardium not available by other imaging modalities. This one test allows for the assessment of ventricular and valvular function, ischemic and nonischemic cardiomyopathies, congenital heart disease, and cardiac tumors. It has been coined by many as "one-stop shopping." As with any imaging modality, it is important to understand not only the indications of the modality but also the patient's perspective and contraindications.

[Simultaneous multiple myeloma and giant cell arteritis without systemic amyloidosis]. / Mieloma multiple y arteritis de celulas gigantes sin amiloidosis.

Primary systemic amyloidosis with clinical and histopathologic features of giant cell arteritis has already been described. The association of multiple myeloma (with primary amyloidosis) and giant cell arteritis is also known. We present the first case in the literature of a patient with multiple myeloma and giant cell arteritis without systemic amyloidosis, suggesting a pathogenic relationship between the two diseases.

Mieloma múltiple y arteritis de células gigantes sin amiloidosis / Simultaneous multiple myeloma and giant cell arteritis without systemic amyloidosis

La amiloidosis sistémica primaria y el mieloma múltiple con amiloidosis primaria se han presentado con características clínicas e histopatológicas que simulan una arteritis de células gigantes. Hasta el momento la asociación se basaba en el rol antigénico del depósito de amiloide sobre las arterias, desencadenando la respuesta inmune que finaliza con una arteritis. Presentamos el primer caso en la literatura de un paciente con mieloma múltiple y arteritis de células gigantes sin amiloidosis sistémica, sugiriendo una relación patogénica entre estas dos entidades. En el caso de nuestro paciente se descartó la presencia de amiloide en la pared arterial, por lo que proponemos que el estímulo para el desarrollo de la arteritis podría ser una excesiva producción de interleuquina 6 fabricada por las células mielomatosas (AU)

Primary systemic amyloidosis with clinical and histopathologic features of giant cell arteritis has already been described. The association of multiple myeloma (with primary amyloidosis) and giant cell arteritis is also known. We present the first case in the literature of a patient with multiple myeloma and giant cell arteritis without systemic amyloidosis, suggesting a pathogenic relationship between the two diseases (AU)

Mieloma múltiple y arteritis de células gigantes sin amiloidosis / Simultaneous multiple myeloma and giant cell arteritis without systemic amyloidosis

La amiloidosis sistémica primaria y el mieloma múltiple con amiloidosis primaria se han presentado con características clínicas e histopatológicas que simulan una arteritis de células gigantes. Hasta el momento la asociación se basaba en el rol antigénico del depósito de amiloide sobre las arterias, desencadenando la respuesta inmune que finaliza con una arteritis. Presentamos el primer caso en la literatura de un paciente con mieloma múltiple y arteritis de células gigantes sin amiloidosis sistémica, sugiriendo una relación patogénica entre estas dos entidades. En el caso de nuestro paciente se descartó la presencia de amiloide en la pared arterial, por lo que proponemos que el estímulo para el desarrollo de la arteritis podría ser una excesiva producción de interleuquina 6 fabricada por las células mielomatosas (AU)

Primary systemic amyloidosis with clinical and histopathologic features of giant cell arteritis has already been described. The association of multiple myeloma (with primary amyloidosis) and giant cell arteritis is also known. We present the first case in the literature of a patient with multiple myeloma and giant cell arteritis without systemic amyloidosis, suggesting a pathogenic relationship between the two diseases (AU)

Mieloma múltiple y arteritis de células gigantes sin amiloidosis / Simultaneous multiple myeloma and giant cell arteritis without systemic amyloidosis

La amiloidosis sistémica primaria y el mieloma múltiple con amiloidosis primaria se han presentado con características clínicas e histopatológicas que simulan una arteritis de células gigantes. Hasta el momento la asociación se basaba en el rol antigénico del depósito de amiloide sobre las arterias, desencadenando la respuesta inmune que finaliza con una arteritis. Presentamos el primer caso en la literatura de un paciente con mieloma múltiple y arteritis de células gigantes sin amiloidosis sistémica, sugiriendo una relación patogénica entre estas dos entidades. En el caso de nuestro paciente se descartó la presencia de amiloide en la pared arterial, por lo que proponemos que el estímulo para el desarrollo de la arteritis podría ser una excesiva producción de interleuquina 6 fabricada por las células mielomatosas

Primary systemic amyloidosis with clinical and histopathologic features of giant cell arteritis has already been described. The association of multiple myeloma (with primary amyloidosis) and giant cell arteritis is also known. We present the first case in the literature of a patient with multiple myeloma and giant cell arteritis without systemic amyloidosis, suggesting a pathogenic relationship between the two diseases

Analysis of engraftment, graft-versus-host disease, and immune recovery following unrelated donor cord blood transplantation.

Unrelated cord blood (UCB) is being used as a source of alternative hematopoietic stem cells for transplantation with increasing frequency. From November 1994 to February 1999, 30 UCB transplant procedures were performed for both malignant and nonmalignant diseases in 27 children, aged 0.4 to 17.1 years. Patients received either HLA-matched (n = 3) or 1- or 2-antigen-mismatched (n = 27) UCB following 1 of 2 standardized preparative and graft-versus-host disease regimens (hyperfractionated total body irradiation, cyclophosphamide, and antithymocyte globulin [ATG] with cyclosporine A and methotrexate; or busulfan, melphalan, and ATG with cyclosporine A and prednisone). The median time to neutrophil and platelet engraftment was 27 days (12-60 days) and 75 days (33-158 days) posttransplantation, respectively. No correlation was noted between neutrophil and platelet engraftment and nucleated cells per kilogram, CD34(+) cells per kilogram infused, or cytomegalovirus status of recipient. The cumulative probability of acute grade 2 or greater graft-versus-host disease (GVHD) was 37.2%, and of grade 3 or greater GVHD was 8.8%. No patients developed chronic GVHD. CD4, CD19, and natural killer cell recovery was achieved at a median of 12, 6, and 2 months, respectively. CD8 recovery was delayed at a median of 9 months. Normal mitogen response was achieved at 6 to 9 months. The probability of survival, disease-free survival, and event-free survival at 1 year was 52.3% (34.1%-70.5%), 54.7% (34.5%-74.9 %) and 49.6% (29.9%-69.4%), respectively. This series of 30 UCB transplants suggests that although CD8 cell recovery is delayed, the pattern of immune reconstitution with UCB is similar to that reported for other stem cell sources. (Blood. 2000;96:2703-2711)

Simultaneous ex vivo expansion of cytomegalovirus and Epstein-Barr virus-specific cytotoxic T lymphocytes using B-lymphoblastoid cell lines expressing cytomegalovirus pp65.

Cytomegalovirus (CMV) infection and Epstein-Barr virus (EBV)-induced lymphoproliferative disease are serious complications associated with allogeneic stem cell transplantation. Immunotherapy using ex vivo expanded, virus-specific cytotoxic T lymphocytes (CTL) has been explored and proven to be effective in therapeutic or prophylactic regimens for CMV and EBV infections. To generate CTL specific for both CMV and EBV, we engineered EBV-transformed B-lymphoblastoid cell lines (BLCL) to express CMV pp65 for use as antigen-presenting cells (APC). BLCL were transduced with a recombinant retrovirus encoding pp65, the immunodominant CMV polypeptide. Western blot analysis and immunocytochemistry confirmed the expression of pp65 in the transduced cells. Peripheral blood mononuclear cells (PBMC) from healthy CMV seropositive donors were stimulated with autologous pp65-expressing BLCL weekly for 3 weeks. Chromium release assays showed that the resulting CTL cultures possessed specific cytotoxicity against EBV and CMV. Recombinant vaccinia viruses encoding individual CMV peptides were used to demonstrate that this CMV-specific cytotoxicity was specific for pp65. Assays on CD4- and CD8-depleted CTL fractions indicated that CD8(+) CTL mediated the pp65-specific cytotoxicity. These CMV/EBV-specific CTL recognized CMV- and EBV-infected targets sharing HLA class I antigens, but not HLA mismatched targets. Our results demonstrate that BLCL can be used as APC to stimulate expansion of EBV- and CMV-specific CTL simultaneously. These findings have potential implications for posttransplant CMV and EBV immunotherapy in recipients of allogeneic stem cell transplants.

CD4(+) Epstein-Barr virus-specific cytotoxic T-lymphocytes from human umbilical cord blood.

Umbilical cord blood (CB) is increasingly used for allogeneic hematopoietic stem cell transplantation. To determine whether viral antigen-specific cytotoxic T-lymphocytes (CTL) could be generated from the predominantly naive T-cell populations in CB, CB-derived mononuclear cells were stimulated with autologous Epstein-Barr virus (EBV) transformed B-lymphoblastoid cell lines over several weeks in the presence of recombinant human interleukin-2 (IL-2). By 28 days of culture, T-lymphocytes from all six CB that had been treated with IL-2 displayed EBV-specific cytotoxicity. These cells were largely CD4(+), with complete inhibition of cytotoxicity by anti-CD3 and variable inhibition by anti-HLA DR monoclonal antibodies. The EBV-specific effectors were cloned by limiting dilution, and most of the CTL clones were CD4(+). The cytotoxicity of the CB-derived CD4(+) CTL clones was inhibited by EGTA but not by anti-Fas ligand mAb, suggesting that this cytotoxicity was mediated by perforin/granzyme B. These data indicate that virus-specific CTL can be cultivated and cloned from CB, a human T-cell source that may not have prior in vivo antigenic exposure or reactivity. This finding may have applications in adoptive immunotherapy to recipients of CB transplants.