Drug-Induced Lung Injury in a Liver Transplant Patient Treated With Sofosbuvir.

New direct-acting antivirals (DAAs) have dramatically improved sustained virologic response (SVR) rates in patients treated for chronic hepatitis C. Although the safety of these agents has been very good in registration trials, unexpected side effects have been reported after much broader use of DAAs on marketing. We retrospectively examined all liver transplant recipients with chronic hepatitis C that received sofosbuvir-based regimens at our clinic. A total of 24 liver transplant recipients with recurrent chronic hepatitis C had received sofosbuvir up to April 2015. Regimens were as follows: sofosbuvir+simeprevir (8), SOF+ledipasvir (6), sofosbuvir+daclatasvir (5) and sofosbuvir+ribavirin (5). Overall, treatment was very well tolerated with only mild adverse events in 42% of patients. However, a 52-year-old woman developed severe respiratory failure within 10 days after beginning sofosbuvir+daclatasvir. High-resolution computerized tomography showed areas of diffused ground-glass opacities in both lungs, suggesting drug-induced lung injury. The bronchoalveolar lavage showed marked signs of acute inflammation without recovering any infectious agent. The patient was treated with high-dose corticosteroids and steadily recovered. DAA therapy was not discontinued, but sofosbuvir was replaced by simeprevir. She reached sustained virologic response after completing 24 weeks of DAA therapy. Given the close temporal association, radiologic and bronchoalveolar lavage findings, and negative work-up for infectious agents, we postulated that sofosbuvir was the most likely explanation for drug-induced lung injury in our patient.

TT genotype of transforming growth factor beta1 +869C/T is associated with the development of chronic kidney disease after liver transplantation.

BACKGROUND: Chronic kidney disease (CKD) is a frequent complication in patients with liver transplantation (LT), and calcineurin inhibitor chronic nephrotoxicity, mediated by transforming growth factor beta1 (TGF-ß1) is an important contributing factor. The aim of this study was to assess the influence of genetic polymorphisms of TGF-ß1 in the development of CKD at 6 months after transplantation. METHODS: One hundred sixty-four LT patients (63.4% male; overall mean age, 48.7 ± 11.6 years) were included in the analysis. CKD was considered at the 6th month after LT and was defined as an estimated glomerular filtration rate (eGFR) of <60 mL/min/1.73 m(2) as calculated on the basis of Modification of Diet in Renal Disease 4-variable equation. TGF-ß1 +869 C/T and +915 G/C polymorphisms were analyzed with the use of hybridization with fluorescent probes and analysis by means of flow cytometry with the Luminex system. The association between the presence of CKD at 6 months and these polymorphisms, as well as with other known risk factors for CKD after LT, was considered. RESULTS: In the univariate analysis, the TT genotype of TGF-ß1 +869 (P = .036; odds ratio, 2.1; 95% confidence interval, 1.1-4.2), age at LT (P < .001), pre-transplantation serum creatinine levels (P = .03), eGFR (P < .001), CKD (P = .027), and immunosuppression with cyclosporine (P = .017) were associated with CKD at 6 months after transplantation. In the multivariate analysis, TGF-ß1 +869TT genotype (P = .017), immunosuppression with cyclosporine (P = .002), age at LT (P = .024), and pre-transplantation CKD (P < .001) remained as independent variables associated with the development of CKD at 6 months after transplantation. CONCLUSIONS: The genetic polymorphism TGF-ß1 +869 C/T may be an independent risk factor for CKD after liver transplantation.

Long-term survivors after pediatric liver transplatation are at increased risk for development of cardiovascular disease events: analysis of 30 cases.

BACKGROUND: Liver transplantation (LT) in adult patients is associated with a higher incidence of cardiovascular risk factors (CVRF), chronic kidney disease (CKD), and cardiovascular disease mortality than the general population. Available information about these problems in adult patients with LT from a pediatric age is limited. The aim of this study was to analyze the incidence of CVRF, risk of developing CKD, and risk of 10-year coronary event in adult patients who received LT in childhood. METHODS: Thirty adult patients (11 female, 19 male; mean age, 29.3 years) who underwent LT in childhood were analyzed, and CVRF, estimated glomerular filtration rate, and current immunosuppressive regimen were recordered. The risk of 10-year coronary event was calculated with the use of validated equations (Framingham and Regicor) and compared with the estimated risk in the general population. RESULTS: None of the patients had CVRF before LT, except 1 patient who received a transplant because of familial hypercholesterolemia. Median age of patients at the time of study was 28.6 years (range, 19.3-43.1 y), and mean follow-up after LT was 17.83 ± 5.21 years. Twenty-nine patients (96.7%) were receiving a calcineurin inhibitor (69% tacrolimus, 31% cyclosporine), along with steroids in 13 of them. The average CVRF per patient was 2, and 11 patients (43.33%) had ≥3. Thirteen patients (43.33%) had CKD. The estimated risk of developing a coronary event at 10 years according to the Framingham score was 3%, higher than expected in the general population of same age and sex. With the use of the Regicor equation, adapted to the Spanish population, the estimated cardiovascular risk was 1.6%, corresponding to Spanish men without CVRF aged 50-55 years. None of the patients had cardiovascular events during the follow-up. CONCLUSIONS: Our data show a high incidence of CVRF and CKD in young adults who received LT in childhood, resulting in an increased risk of cardiovascular disease.

CC genotype at rs12979860 of IL28B is associated with lower risk of new-onset diabetes after transplantation in adult patients with liver transplantation for hepatitis C cirrhosis.

INTRODUCTION: New-onset diabetes mellitus after transplantation (NODAT) in patients undergoing liver transplantation (LT) for hepatitis C virus (HCV)-related cirrhosis is associated with more aggressive HCV recurrence on the graft, rapid progression of fibrosis, and lower rate of sustained viral response to antiviral therapy. The CC genotype at rs12979860 of the IL28B is associated with greater rates of spontaneous clearance of HCV and response to antiviral therapy. IL28B acts on the interferon-stimulated genes through the JAK-STAT pathway, which is related to the development of insulin resistance. The aim of this study was to investigate whether IL28B rs12979860 polymorphism is associated with the development of NODAT after LT for cirrhosis owing to HCV infection. METHODS: We analyzed 99 patients (age, 52.7 ± 9.4 years; 70% male) who underwent LT for HCV-related cirrhosis, with ≥1 year of follow-up and with available DNA sample. NODAT was defined starting from the sixth month after LT, according to the international consensus guidelines. Genotyping was carried out by real-time polymerase chain reaction and analysis of the melting temperature with the LightCycler 480 system. RESULTS: Twenty-eight patients (28.3%) developed NODAT. CC genotype at rs12979860 of IL28B was associated with a lesser incidence of NODAT versus non-CC genotypes (P = .05; odds ratio, 0.31; 95% CI, 0.11-0.92). We did not find any association between NODAT and age at transplantation, gender, pretransplant body mass index, presence of hepatocellular carcinoma, type of initial immunosuppression (cyclosporine, tacrolimus or corticosteroids) or acute rejection treated with steroids. CONCLUSION: The CC genotype at rs12979860 of IL28B is a protective factor for NODAT in patients with LT for HCV-related cirrhosis.

Toll-like receptor 3 L412F polymorphism may protect against acute graft rejection in adult patients undergoing liver transplantation for hepatitis C-related cirrhosis.

Liver transplantation activates the innate immune system by toll-like receptors (TLRs), potentially leading to allograft rejection and graft failure. The aim of this study was to evaluate the possible association of different single nucleotide polymorphisms (SNPs) in several TLR genes with the incidence of acute graft rejection in liver transplant recipients for hepatitis C virus (HCV)-related cirrhosis. This is a single-center study of 100 adult patients who received a first whole only liver graft from deceased donors at our institution between 1988 and 2009 for cirrhosis due to HCV infection. We examined 10 SNPs in the TLR1 (S6021), TLR2 (R753Q), TLR3 (L412F), TLR4 (D299G and T399I), TLR5 (R392X), TLR6 (S249P), TLR7 (Q11L), and TLR9 (-1237T/C and -1486C/T) genes. Genotyping was carried out with the LightSNiP typing assay (TIB-MolBiol, Berlin, Germany) by analyzing the melting curves with the LightCycler 480 system (Roche Applied Science, Mannheim, Germany). Recipient allelic and genotypic distributions for each SNP were compared among patients with and without acute rejection within the first 3 months after transplantation. We found the homozygous mutant TT genotype for TLR3 L412F was associated with a lower rate of acute rejection when compared with the homozygous wild-type genotype [odds ratio (OR) = 0.1, 95% confidence interval (95% CI) = 0.01-0.86; P = .017], and showed a trend toward a lower graft rejection rate when compared with patients carrying one or two C alleles (OR = 0.15, 95% CI = 0.02-1.2, P = .05). No other associations with acute rejection rates were found for any other SNP evaluated. This preliminary study suggests an important role for SNP TLR3 L412F in acute rejection in liver transplant patients for HCV-related cirrhosis. Nevertheless, these findings must be prospectively validated in other cohorts of patients as well as in patients after liver transplantation for other etiologies than HCV.

Heat shock protein 70 and cellular disturbances in cochlear cisplatin ototoxicity model.

BACKGROUND: Exposure to cisplatin leads to cochlear cell death by apoptosis; these changes are most marked on the seventh day after exposure. Heat shock proteins are induced in inner ear cells in response to a variety of stimuli. This study examined the role of heat shock protein 70 in cisplatin-induced cochlear cell death. METHODS: Fifty-six Sprague-Dawley rats were involved. Some were injected with cisplatin (5 mg/kg body weight), some with cisplatin plus the caspase inhibitor Z-Asp(OMe)-Glu(OMe)-Val-Asp(OME)-fluoromethylketone (5 mg/kg body weight) and others were left as controls (being injected only with saline). Seven days later, we examined the expression of heat shock protein 70 and several other apoptosis-related proteins within the rat cochlear cells; we also assessed total superoxide dismutase activity, auditory brainstem response and auditory steady state response. RESULTS: Seven days after cisplatin injection, significantly increased expression of heat shock protein 70 was found within the rat cochleae. This correlated with increased executioner caspase levels, total superoxide dismutase activity and auditory brainstem response thresholds, and a significant elevation in auditory steady state response thresholds. Inhibition of caspase-3 activity significantly reduced cochlear heat shock protein 70 expression and total superoxide dismutase activity, and improved auditory brainstem response and auditory steady state response thresholds. CONCLUSIONS: Seven days after cisplatin exposure, we found disturbances of the cochlear cellular machinery involving heat shock protein 70, other apoptotic proteins and total superoxide dismutase.

Interleukin-10 promoter polymorphisms in patients with systemic lupus erythematosus from the Canary Islands.

The purpose of this study was to examine whether several allelic variants in the polymorphic interleukin (IL)-10 promoter region were related with an increased risk of developing systemic lupus erythematosus (SLE) in Spanish patients from Canary Islands. Microsatellites (MS) at positions -4000 and -1200 (IL10R and IL10G, respectively) and single nucleotide polymorphisms (SNPs) (MS) at positions -1082G/A, -819C/T and -592C/A of the IL-10 promoter were analysed in patients with SLE and healthy controls from Canary Islands (Spain). We found that SNPs but not MS were associated with SLE. The GCC haplotype frequency was significantly higher in SLE patients (0.43) than in healthy donors (0.33) [P = 0.02; OR = 1.50 (95% CI = 1.06-2.14)], whereas the ACC haplotype was less represented in patients (0.28 vs. 0.37) [P = 0.02; OR = 0.64 (95% CI = 0.44-0.92)]. To assess the functional role of genotypes, serum IL-10 levels from patients and controls were quantified by ELISA. Also, the lipopolysaccharide-induced IL-10 secretion by monocytes from healthy controls was evaluated in vitro. Serum IL-10 levels were higher in patients [median (interquartile range) = 2.8 pg/mL (1.8-4.2)] than in controls [0.9 pg/mL (0-3.5)] (P = 0.02), but no association was observed between serum IL-10 levels or lipopolysaccharide-induced IL-10 secretion and the IL-10 promoter haplotypes. These data suggest that the IL-10 promoter haplotype that produces higher levels of cytokine is associated with SLE in patients from Canary Islands.

HSP-70 as a nonspecific early marker in cisplatin ototoxicity.

CONCLUSION: The great variety of pathological entities related to the presence of circulating HSP-70 suggests a nonspecific cellular damage. As the present study shows, positive results decrease with respect to the time elapsed after the injection of the ototoxic agent. HSP-70 appears as an early and transient marker that could permit early detection of inner ear damage. OBJECTIVES: The aim of this study was to determine the presence of HSP-70 at different time points by means of Western blot immunoassay in the sera of rats treated with cisplatin. MATERIALS AND METHODS: Thirty-six Wistar rats were intraperitoneally injected with cisplatin at a dose of 5 mg/kg and blood samples were collected at 7 and 90 days. Determination of HSP-70 was made by means of a modified Western blot immunoassay kit originally used for human HSP-70 antigen detection. A control group of 18 animals was used for comparison. RESULTS: Western blot was positive in 77.8% of the animals in the 7 days group, decreasing to a 44.4% in the 90 days group. In the control group, Western blot was positive in 5.5%.

The presence of an intronic deletion in p73 and high levels of ZEB1 alter the TAp73/DeltaTAp73 ratio in colorectal carcinomas.

TAp73 variants largely mimic p53 suppressor activities, while DeltaTAp73 forms act as oncogenes through the inactivation of p53 and TAp73. The present study analysed how TAp73 and DeltaTAp73 levels might be affected by the presence of a 73 bp deletion in a regulatory region of p73. The clinical relevance of this deletion was also examined. ZEB1 can bind to the region repressing p73 transcription in vitro. The relationship between ZEB1 and p73 variant expression levels was studied in the context of this deletion and the levels of the ZEB1 cofactors p300 and CtBP. Tumour and normal tissue from 81 colorectal cancer patients was analysed to evaluate firstly the levels of TAp73, DeltaTAp73 (DeltaEx2p73, DeltaEx2/3p73, and DeltaNp73), ZEB1, p300, and CtBP by quantitative real-time RT-PCR, and secondly the presence of the 73 bp deletion. Tumour characteristics were examined in each patient. Suppressor and oncogenic isoforms of p73 were co-up-regulated in tumour tissues. Overexpression of p73 variants was associated with adverse tumour features. The 73 bp deletion was present in 40% of the patients and was associated with adverse pathological parameters of the tumours and also with TAp73 down-regulation. In those cases harbouring the deletion, the levels of ZEB1 and those of DeltaEx2p73, DeltaEx2/3p73, and DeltaNp73 correlated directly. Variations in the concentration of p300 affected the observed correlations between ZEB1 and the different p73 variants. In conclusion, in colorectal cancer, the 73 bp deletion in the first intron of the p73 gene and different expression levels of ZEB1 and p300 may act in concert to affect the ratio of TAp73/DeltaTAp73 forms, favouring p73 oncogenic variants. In addition, up-regulation of p73 oncogenic isoforms predicts a poor prognosis based on its relationship with advanced tumour stage.

The dinucleotide repeat polymorphism in the 3'UTR of the CD154 gene has a functional role on protein expression and is associated with systemic lupus erythematosus.

OBJECTIVE: To investigate the association of the (CA)n dinucleotide repeat in the 3' untranslated region (3'UTR) of the CD154 gene with systemic lupus erythematosus (SLE), and its functional role in protein expression. METHODS: The allelic and genotypic distributions of the polymorphism were compared in 80 patients with SLE and 80 controls. A complete clinical and analytical database was recorded in each patient in order to correlate the clinical manifestations in SLE with different alleles. To investigate the functional role of the polymorphism, the CD154 protein expression on activated lymphocytes from healthy homozygous controls was evaluated by flow cytometry. RESULTS: The 24 CA allele was the most represented in controls (p = 0.029), whereas the alleles containing >24 CA repeats were found in patients (p = 0.0043). Furthermore, when only homozygous women were considered, most controls carried two 24 CA alleles (p = 0.041), whereas most patients carried two alleles containing >24 CA repeats (p = 0.032). Also, patients carrying at least one 24 CA allele had less neurological involvement (p = 0.034), and carriers of at least one allele with fewer than 24 CA repeats presented more livedo reticularis (p = 0.006) and anti-Sm (p = 0.01) and anti-RNP (p = 0.038) autoantibodies. CD154 maximum expression in activated lymphocytes from all controls was reached after 54 hours, but it was more prolonged in controls carrying two alleles with >24 CA repeats (p = 0.0068). CONCLUSION: The CD154 3'UTR microsatellite is associated with SLE, and the most represented alleles in patients were accompanied by a more prolonged protein expression in activated lymphocytes from controls.

Production of intracellular IL-2, TNF-alpha, and IFN-gamma by T cells in B-CLL.

BACKGROUND: Recent evidence indicates that the slowly expanding population of CD5(+) B cells that characterizes B-cell chronic lymphocytic leukemia (B-CLL) could be related to defects in the response to cytokine produced by T cells that regulate apoptosis. We studied the intracellular expressions of interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) in T-helper 1 cells (Th1 response) of B-CLL. METHODS: Peripheral blood mononuclear cells from 21 healthy individuals and purified T cells from 21 early-stage and 15 late-stage B-CLL patients were activated with phorbol myristate acetate and ionomycin. The Th1 cytoplasmic cytokines were evaluated in CD4(+) and CD8(+) T cells by flow cytometry. RESULTS: The percentages of CD4(+) and CD8(+) T cells positive for IL-2 were significantly lower in B-CLL patients than in healthy individuals (P = 0.030 and 0.049, respectively). No significant differences in TNF-alpha or IFN-gamma intracellular expressions were found between patients and healthy individuals. TNF-alpha- and IFN-gamma-expressing CD8 T cells were disease stage dependent, being significantly higher in late-stage patients (P < 0.001 for both cytokines). CONCLUSIONS: Our present observations suggested that Th1 cytokines may be of major importance in the pathogenesis of B-CLL.

CD154 polymorphism in Spanish populations. Differences in the allelic distribution between Canary islanders and Peninsulars.

The CD154 gene contains a dinucleotide repeat (CA)n in the 3' untranslated region. Allelic distribution in Spanish populations from two areas with different genetic background, the Canary Islands and Peninsula, are described. Seven alleles with different allelic distribution between the two groups, were found. This represents a highly polymorphic marker, useful for genetic studies on a critical molecule in immunity.

SPECA: a program for the calculation of thermodynamic equilibrium constants from spectrophotometric data.

A computer program called SPECA and written in compiled BASIC (Microsoft QuickBasic Ver. 4.5) has been written for the calculation of thermodynamic constants in solution equilibria studies from spectrophotometric measurements. In order to calculate the thermodynamic constants it is necessary to introduce different theories to estimate the activity coefficients of the system involved under investigation. In this version of the program the equations proposed in the models of Debye-Hückel. Specific Interaction Theory (SIT) or the Modified's Bromley Theory can be chosen. To achieve its purpose, the program uses absorbance and activity or free concentration data of one of the components, normally H(+), and it is not limited with respect to the number of components that can be studied. Minimization of the error square sum in the absorbance, which can be absolute or relative, is carried out using the Levenberg-Marquardt-Nash algorithm at three different levels: equilibrium constants in the first level, molar absorptivities in the second and interaction coefficients in the third for those systems using the SIT or the Modified Bromley's Theories.

Automation of Vapour Pressure Osmometry measurements.

A program has been developed for the control of Vapour Pressure Osmometry (VPO) measurements. The output signal of a Vapour Pressure Osmometer is read by an A/D converter card installed in one of the expansion slots of a PC microcomputer. The stability of the measurements is checked by analysing the first derivative of the smoothed signals, which is calculated in real time. Sets of repeated measurements are carried out under the supervision of a computer program as a check for their reproducibility. When the set is ended the program calculates the average and its standard deviation.The program is particularly valuable for evaluating VPO measurements for solution equilibria studies. This interfacing strategy may be applied to any kind of technique in which the time stability of the signals is the basis for defining measurement stability. The automated VPO has other advantages, including low cost and time saving.