Analysis of INF-gamma, TNF-alpha and dendritic cells to predict hepatitis C virus recurrence in liver transplant patients.

INTRODUCTION: Hepatitis C virus (HCV) infection is one of the leading causes of chronic liver disease and the reason for more than 50% of liver transplantations (OLT). Recurrent HCV infection occurs in almost all transplant recipients and has an unfavorable course. Although immunosuppressive agents are necessary to avoid allograft rejection, these drugs may favor viral replication facilitating viral-mediated graft injury. METHODS: To predict the evolution of two HCV(+) patients who underwent OLT, we studied INF-gamma and TNF-alpha production and the maturation capacity of dendritic cells (DCs) at three time points: before transplantation (Pre-Tx) and at 2 (2M) and 6 (6M) months after transplantation. Cytometric bead assays were used to quantify INF-gamma and TNF-alpha production in the supernates of mixed leukocyte reactions (MLR) between spleen cells from the liver donor and CD4(+) cells from the recipients. Immature and mature DCs were generated in vitro from patient monocytes. RESULTS: The one patient who experienced recurrent HCV showed loss of CD4(+) responses to donor antigens and INF-gamma and TNF-alpha production after OLT. In contrast, the other patient maintained detectable levels of these cytokines after OLT. It was possible to generate mature DCs from monocytes with the aid of CD40L in both cases, but decreased expression of HLA-DR, CD80, and CD86 markers was observed upon posttransplantation analyses in the patient with recurrent HCV. CONCLUSION: Loss of the proliferative response as well as INF-gamma and TNF-alpha production, together with a decreased HLA-DR, CD80, and CD86 (markers of mature DCs), indicated an inadequate immune response to viral progression in the liver transplant recipient with relapsing HCV infection.

Leukocyte Reduction Filters: an alternative source of Peripheral Blood Mononuclear Cells / Filtros de leucodepleción: una alternativa para la obtención de células mononucleares de sangre periférica

The use of human Peripheral Blood Mononuclear Cells (PBMC) for research purposes is widely extended. Typically, PBMC are separated from blood donations after volume reduction (buffy coat) and a subsequent density gradient centrifugation. However, policies on blood donation and processing for transfusion medicine have introduced the use of Leukocyte Reduction Filters (LRF). Blood Transfusion Services still process blood samples and produce buffy coats, but only a few units and specifically to obtain random platelets. Therefore, it is important to find alternative sources of PBMC. We have tested the possibility to use the cells retained by the LRF as an alternative source, with special interest on monocytes, because of their potential use as dendritic cell (DC) precursors for immunotherapy. Cells from filters were recovered at high numbers (about 5x108 PBMC) and viability (> 95%), and the phenotypic and functional experiments confirmed that they were similar to those cells obtained from buffy coats. Monocytes were differentiated in vitro to DC, showing no differences from those obtained from the buffy coats. The results have shown that elution of PBMC from LRF is a valid alternative source to obtain all blood cell types, including monocytes to generate functional DC (AU)

Las células mononucleares de sangre periférica (PBMC, Peripheral Blood Mononuclear Cells) son de uso común en los laboratorios de investigación. En general, las PBMC se obtienen a partir de donaciones de sangre - de las que se obtienen los «buffy coats» (BC)– y tras un proceso de centrifugación en gradiente de densidad. Sin embargo, en los últimos años se ha introducido el uso de los filtros de leucodepleción en el procesamiento de la sangre destinada a la medicina transfusional. Los Bancos de Sangre aún procesan y producen BC, en general para obtener «pools» plaquetarios, aunque la tendencia es a su desaparición. Por ello, es importante definir fuentes alternativas de las que obtener los PBMC. Una posibilidad, que hemos querido comprobar, es la utilización de las células que quedan retenidas en los filtros, en especial de monocitos, debido a su potencial uso como precursores de células dendríticas para inmunoterapia. Los resultados que hemos obtenido muestran que las células pueden ser eluidas de los filtros en un elevado número (del orden de 5x108 PBMC) y viabilidad (>95%). Además, el perfil fenotípico y funcional es similar al obtenido al analizar células generadas a partir del clásico BC. Los monocitos se diferenciaron in vitro a células dendríticas, de forma similar a los monocitos obtenidos a partir de BC. En resumen, la elución de las células retenidas en los filtros de leucodepleción es una alternativa válida a los clásicos BC para obtener todos los tipos celulares presentes en sangre periférica, incluyendo monocitos para su diferenciación en células dendríticas (AU)

Feasibility trial of methotrexate-paclitaxel as a second line therapy in advanced urothelial cancer.

To evaluate the clinical value of the concurrent use of methotrexate administered immediately before paclitaxel, we investigated the efficacy and toxicity of this two-drug combination administered as palliative second line therapy in patients with advanced urothelial cancer. The design of the schedule and sequence used was based on our previous preclinical data from a comparative study on sequential combinations of paclitaxel and methotrexate in a human bladder cancer cell line. As a confirmation study, we further extended our analysis of in vitro synergism. Twenty patients with advanced transitional cell carcinoma of the urinary tract previously treated with platinum-based therapy, with adequate renal function and a performance status > or = 60 were considered eligible. They received therapy with methotrexate 30 mg/m2 administered as an intravenous bolus, followed immediately by paclitaxel 175 mg/m2 given as a 3-hr infusion, both on day 1 every 21 days. Therapy was given on a compassionate-use basis until either disease progression was documented or the patient became intolerant to therapy. In vitro data were further analyzed using the median-effect principle and the combination index method. Twenty patients with metastatic (16 patients) or locally advanced disease (four patients) received a median of three cycles of therapy. Of the 19 patients assessable for response, there were six partial responses and seven disease stabilizations with no complete responses. Median duration of response was 3 months (range, 2-7) and median survival was 5 months. Three patients developed grade 3-4 neutropenia, one patient had grade 3 anemia, four patients had grade 2-3 sensory neuropathy, and three patients had myalgias. Eighteen patients developed alopecia. Gastrointestinal toxicity was mild. One patient died after the first cycle due to pulmonary thrombo-embolism and could not be evaluated for response. The synergistic in vitro effect of the concurrent combination was confirmed in analyses performed under mutually exclusive and mutually nonexclusive criteria. In conclusion, the combination of methotrexate and paclitaxel at this dose and sequence is feasible and active as a palliative therapy in patients with advanced urothelial cancer previously treated with platinum-based therapies. This schedule merits further investigation in a phase-II trial.

The obesity hypoventilation syndrome can be treated with noninvasive mechanical ventilation.

STUDY OBJECTIVES: To assess the effectiveness of nasal noninvasive mechanical ventilation (NIMV) in patients with obesity hypoventilation syndrome (OHS). DESIGN: Clinical assay that compares two groups of patients with hypercapnic respiratory failure, one group with OHS and the other group with kyphoscoliosis, in their basal situation and after 4 months of treatment with nocturnal NIMV. Thirty-six patients (22 patients with OHS and 14 patients with kyphoscoliosis) completed the study protocol. RESULTS: The frequency of symptoms, such as morning headache, morning drowsiness, dyspnea, and leg edema, improved in a statistically significant way in both groups of patients. The sleepiness improved only in the group with OHS. The comparison of frequency of symptoms between both groups of patients after NIMV treatment did not present statistically significant differences. In the resting situation and without nasal ventilation in place, the PO(2) (mean +/- SD) changed from 51 +/- 10 to 64 +/- 11 mm Hg (p < 0.001) and PCO(2) from 58 +/- 10 to 45 +/- 5 mm Hg (p < 0.001) when the patients with OHS were treated with NIMV. In the group of patients with kyphoscoliosis, likewise without nasal ventilation in place, PO(2) changed from 53 +/- 6 to 65 +/- 5 mm Hg (p < 0.001) and PCO(2) from 59 +/- 11 to 45 +/- 4 mm Hg (p < 0.001) with NIMV treatment. When we compared PO(2) and PCO(2) in both groups of patients at the beginning and at the end of NIMV treatment, we did not find statistically significant differences between OHS and kyphoscoliosis. CONCLUSIONS: NIMV improves the clinical symptoms and the respiratory failure of patients with OHS to a similar degree to that reported for diseases in which its use is completely established, such as kyphoscoliosis. Therefore, NIMV could be an alternative to the treatment of patients with OHS.

Comparative study of sequential combinations of paclitaxel and methotrexate on a human bladder cancer cell line.

We investigated the cytotoxic effect of different sequential combinations of paclitaxel (Taxol, TX), an antimicrotuble agent, and methotrexate (MTX), a potent inhibitor of dihydrofolate reductase, on a human bladder cancer cell line (T24). The results obtained with a colony-forming assay 5 days after 24 hr of exposure to TX alone showed a strong decline in colony growth up to a concentration of 6 nM. There was a plateau phase at TX concentrations ranging from 6 to 10 nM. The IC50 values found with the different dosing sequences and 24-hr exposure to the drugs were 0.24 microM for MTX alone, 3.49 nM for TX alone, 3.84 nM for TX followed by MTX, 3.75 nM for TX and MTX simultaneously, and 2.10 nM for MTX followed by TX. The cytotoxic effect due to drug interactions in the different sequential combinations was evaluated by an algebraic method. All the combination sequences were found to be synergistic (combination index [CI] < 1) at low TX concentrations. An antagonistic effect (CI > 1) was obtained at higher TX concentrations. Our data demonstrate that the T24 cell line is sensitive to TX and that the cytotoxic effect is enhanced to different degrees depending on the treatment sequence when TX is combined with MTX. These findings could have implications in the design of appropriate administration schedules of these two drugs for the treatment of bladder cancer.

[Usefulness of transbronchial punction and mediastinoscopy in mediastinal nodal staging of non-microcytic bronchogenic carcinoma. Preliminary study]. / Utilidad de la punción transbronquial y la mediastinoscopia en la estadificación ganglionar mediastínica del carcinoma broncogénico no microcítico. Estudio preliminar.

Preliminary study to compare the sensitivity and specificity of transbronchial needle aspiration (TBNA) and mediastinoscopy/anterior mediastinotomy (MED/AMED) and/or thoracotomy for staging of mediastinal nodes in non-small cell carcinoma. To determine the sensitivity and specificity of computerized tomography (CT) as a screening technique. Thirty-three patients with non-small cell carcinoma but no remote metastasis and good lung function were evaluated. A chest CT scan was performed before bronchoscopy in 27 patients and before surgery in the others. Nodular areas considered diseased based on CT images were staged by TBNA. When CT images were not available before bronchoscopy. TBNA for staging was performed in the subcarinal region. Results by TBNA were compared with those obtained by MED/AMED and/or thoracotomy. The prevalence of metastatic nodular disease was 47%. CT detected enlarged mediastinal nodes in 24 patients; the images were considered normal in 9 patients. Sensitivity and specificity of CT was 93% and 54%, respectively, with a positive predictive value (PPV) of 68% and negative predictive value (NPV) of 87.5%. The sensitivity and specificity of MED/AMED were 73% and 100%, respectively; PPV was 100% and NPV was 75%. The sensitivity and specificity of TBNA were 36% and 92%, respectively; PPV was 83% and NPV was 57%. The pneumothorax with pleural empyema suffered by one patient after MED could have been avoided, given that the earlier TBNA was positive. TBNA is a safe, useful technique for staging nodes in non-small cell carcinoma. Although the sensitivity of TBNA is lower than that of MED, regions that are difficult to reach with the latter technique can be sampled by TBNA. Furthermore, MED can be rendered unnecessary by positive TBNA results. CT imaging of the chest is sensitive but its specificity is low for detecting ganglial metastasis.

Noninvasive positive pressure ventilation and not oxygen may prevent overt ventilatory failure in patients with chest wall diseases.

Some patients with chest wall diseases (CWD) without respiratory failure manifest important alterations in nocturnal gas exchange, as a previous stage to the future development of daytime respiratory failure. The purpose of this study was to evaluate the efficacy of nasal intermittent positive pressure ventilation (NIPPV) during sleep in a group of obese patients and in another group with restrictive thoracic diseases (RTD), comparing the results with those obtained from conventional nocturnal oxygen therapy. From a total of 42 patients with CWD free of daytime respiratory failure, 27 (64%) were considered nocturnal oxygen desaturators without sleep apnea and were included in the study. The study protocol was completed by 21 of these patients. After 2 weeks of treatment, symptoms of dyspnea, morning headaches, and morning obnubilation improved significantly (p<0.05) in both groups of patients after NIPPV but not with oxygen. Baseline daytime PaO2 was 68+/-7 mm Hg in the obese group of patients and 73+/-11 mm Hg in the RTD group. It improved significantly with NIPPV to 73+/-5 mm Hg in obese patients (p<0.05) and to 77+/-12 mm Hg in the RTD group (p<0.05) but did not change with oxygen (68+/-8 mm Hg in the obese group and 73+/-12 mm Hg in the RTD group). Both treatments improved oxygen saturation during sleep, but oxygenation tends to be higher with oxygen than with NIPPV. Only NIPPV was able to normalize the baseline nocturnal alveolar hypoventilation. From the 21 patients treated, 19 decided to continue with long-term NIPPV, one with oxygen, and one refused treatment. We conclude that in patients with CWD who manifest nighttime oxygen desaturation and hypoventilation, early initiation of NIPPV is preferable to supplemental oxygen. Our results also suggest that NIPPV initiated before overt ventilatory failure could prevent its onset.

Differences in the binding of blocking anti-CD11b monoclonal antibodies to the A-domain of CD11b.

CD11b/CD18 (Mac-1) is a leukocyte integrin that plays a critical role in neutrophil adhesion and the initiation of acute inflammatory responses. Several Mac-1 blocking mAbs bind to the A-domain of CD11b, a approximately 200 amino acid region in the N-terminal portion of the protein that is involved in ligand binding and Mac-1 functional activity. We examined several CD11b blocking mAbs for different patterns of binding to A-domain. We used human/murine chimeric CD11b expression constructs and deletions of the A-domain to examine binding. We describe the binding characteristics of mAbs 60.1, LM2/1, LPM19C, M170, 44, and 904. All of these mAbs, except for 60.1, bind to the C-terminal half of the human A-domain (CD11b181-316). mAb 60.1 was unique in that it required regions of the N- and C-terminal ends of the A-domain for binding. mAbs 60.1, LPM19C, 904, and 44 all required the A-domain to be intact for binding. This suggests that these CD11b mAbs recognize a conformational epitope. LM2/1 was capable of binding to a fragment of the A-domain, CD11b285-300. Inasmuch as this system has been used to define different mAb binding sites, it may be used to analyze specific ligand binding sites in the A-domain of CD11b.

Colonisation with Aspergillus of an intralobar pulmonary sequestration.

Pulmonary sequestration is a term used to describe an area of embryonic lung tissue supplied by an anomalous systemic artery. Two forms are recognised-extralobar and intralobar-with different clinical presentations. A patient is reported with intralobar pulmonary sequestration in the left lung and colonisation with Aspergillus which was successfully treated by lower lobectomy.

Purine enzyme profile in human colon-carcinoma cell lines and differential sensitivity to deoxycoformycin and 2'-deoxyadenosine in combination.

Different cell lines, 2 from human colon carcinoma (LoVo and HT29) and 1 from Chinese hamster ovary (CHO K-I), were examined to assess the effect of deoxycoformycin (dCF), an inhibitor of adenosine deaminase (ADA), and 2'-deoxyadenosine (dAdo) on their growth. When used alone, neither dCF or dAdo were cytotoxic for the 3 cell lines, while their combination caused inhibition of cell growth, with the following sensitivity: CHO K-I > LoVo > HT29. We studied the pattern of enzymatic activities involved in the metabolism of dAdo in the 3 cell lines. The phosphorylation of dAdo by adenosine kinase appears to play a central role in the toxicity of the deoxynucleoside in combination with dCF. In fact, CHO K-I cells, which are the most sensitive, possess the highest level of this enzyme. Moreover, the cytotoxic effect was almost completely reversed in the 3 cell lines when inhibitors of adenosine kinase, such as 5'-amino-5'-deoxyadenosine and iodotubercidine, were added to the culture medium together with dCF and dAdo. In addition, baby hamster kidney (BHK) adenosine-kinase-deficient (AK-) cells were highly resistant to this treatment. Uptake inhibition of dAdo using dipyridamole also caused reversal of the toxicity. The AMP and deoxyAMP dephosphorylating activities, much lower in the CHO K-I cells, also appear to play a central role in the toxicity of dAdo when adenosine deaminase is inhibited. However, our data suggest that other factors may modulate the toxic effect, such as S-adenosyl-homocysteine-hydrolase inhibition by dAdo at high concentrations.

Squamous cell carcinoma antigen (SCC Ag) in the diagnosis and prognosis of lung cancer.

OBJECTIVE: We have studied the usefulness of squamous cell carcinoma antigen (SCC Ag) in diagnosis and prognosis of lung cancer (LC). MATERIAL AND METHODS: We have measured the serum SCC Ag levels in 388 subjects: 69 healthy persons; 103 with nonmalignant lung diseases (NMLD); 24 with lung metastasis of extrapulmonary origin (LMEO); and 192 with LC (88, with squamous cell carcinoma [SCC] type). In 55 with SCC, we analyzed the survival time. RESULTS: Serum SCC Ag was above 2.5 ng/ml in 1.4 percent of healthy persons; 2.9 percent of those with NMLD; 8.3 percent of those with LMEO; and 27.6 percent of those with LC. Such percentage was 47.7 percent in SCC. In this type, there were significant differences according to the extent of disease (61.6 percent in advanced stages, and 26.5 percent in localized stages, p = 0.002). In the other types, the sensitivity was substantially lower. The initial SCC Ag has prognostic significance (p = 0.02) in the univariate analysis, but it loses such significance in a multivariate model, including the stage. CONCLUSIONS: Therefore, we do not recommend this marker in the clinical management of patients with LC, even it can be useful in the differential diagnosis if used in combination with other markers.

Fibrous polyp of the ureter.

The most frequent ureteral tumors are transitional carcinoma. Benign neoplasms are uncommon. In this paper we present 6 cases of ureteral polyps, which represent 11.1% of all the tumors in our series of ureteral tumors. We have reviewed the possible etiopathogenesis and exposed the clinical characteristics. Secondary to the obstructive phenomenon and the radiologic signs, it is the tigroid appearance, the localization and the absence of total obstruction which allows the diagnostic suspicion. The treatment of choice is partial resection. Ureterostomy with peroperatory biopsy, in cases in which the malignancy is not yet established, is recommended in order to avoid unnecessary nephroureterectomies.

45,X/46,X,dic(Yq) mosaicism and mixed gonadal dysgenesis. Case report and review of the literature.

A 4 year 7 month-old boy with ambiguous genitalia, histological evidence of mixed gonadal dysgenesis, and 45,X/46,X,dic(Yq) mosaicism is reported. The identity of the dicentric Y chromosome was stablished by its typical fluorescent banding patterns and the presence of two centromeres demonstrated by C-band technique. A review of the literature yielded nine additional cases of mosaic 45,X/46,X,dic(Yq). Phenotypical and histological findings among these cases were compared, and the possible localization of the genes responsible for testicle induction and maturation is discussed.