Expression of IFN-λ1 from Congjiang pigs and its effect on anti-PRRSV proliferation.

An eukaryotic expression system of Congjiang pigs IFN-λ1 was constructed to obtain its expression in CHO-K1 cells and the inhibition effect of Congjiang pig IFN-λ1 on PRRSV proliferation was verified. The eukaryotic expression plasmid pEGFP-PoIFN-λ1 was constructed from the pig IFN-λ1 gene fragment and transfected into CHO-K1 cells. Expression was detected by fluorescence microscopy and Western blotting. The influence on the proliferation of PRRSV was assessed. The results of the study showed that the recombinant plasmid pEGFP-PoIFN-λ1 was constructed correctly. After transfection, green fluorescent signal was detected in CHO-K1 cells by fluorescence microscopy. Western blot analysis revealed that in cells at different time periods after transfection, porcine IFN-λ1 was expressed, with the highest expression observed 36 h after transfection. The antiviral activity of the supernatant after 36 h of transfection was determined by the micro cytopathic inhibition method, and the biological activity was 2.1×103 U/mL. Quantitative PCR was used to detect the proliferation of PRRSV, and the results showed that Congjiang pigs IFN-λ1 significantly inhibited the mRNA expression of PRRSV and viral proliferation in a dose- and time-dependent manner. This study established a Congjiang pig IFN-λ1 eukaryotic expression system, and the quantitative PCR method showed that it has a significant inhibitory effect on the proliferation of PRRSV, which lays a foundation for the future production of antiviral drugs and clinical application.

A Double-Blind, Double-Dummy, Flexible-Design Randomized Multicenter Trial: Early Safety of Single- Versus Divided-Dose Rabbit Anti-Thymocyte Globulin Induction in Renal Transplantation.

A previous nonblinded, randomized, single-center renal transplantation trial of single-dose rabbit anti-thymocyte globulin induction (SD-rATG) showed improved efficacy compared with conventional divided-dose (DD-rATG) administration. The present multicenter, double-blind/double-dummy STAT trial (Single dose vs. Traditional Administration of Thymoglobulin) evaluated SD-rATG versus DD-rATG induction for noninferiority in early (7-day) safety and tolerability. Ninety-five patients (randomized 1:1) received 6 mg/kg SD-rATG or 1.5 mg/kg/dose DD-rATG, with tacrolimus-mycophenolate maintenance immunosuppression. The primary end point was a composite of fever, hypoxia, hypotension, cardiac complications, and delayed graft function. Secondary end points included 12-month patient survival, graft survival, and rejection. Target enrollment was 165 patients with an interim analysis scheduled after 80 patients. Interim analysis showed primary end point noninferiority of SD-rATG induction (p = 0.6), and a conditional probability of <1.73% of continued enrollment producing a significant difference (futility analysis), leading to early trial termination. Final analysis (95 patients) showed no differences in occurrence of primary end point events (p = 0.58) or patients with no, one, or more than one event (p = 0.81), or rejection, graft, or patient survival (p = 0.78, 0.47, and 0.35, respectively). In this rigorously blinded trial in adult renal transplantation, we have shown SD-rATG induction to be noninferior to DD-rATG induction in early tolerability and equivalent in 12-month safety. (Clinical Trials.gov #NCT00906204.).

Excellent outcomes can be achieved in young pancreas transplant alone recipients by addition of sirolimus to maintenance immunosuppression regimen.

BACKGROUND: Pancreas transplant alone (PTA) has evolved into a viable treatment option for nonuremic patients with labile diabetes mellitus. Historically, PTA outcomes were inferior to simultaneous pancreas-kidney transplant outcomes, because of the higher rate of graft loss due to rejection in PTA recipients. But with advances in immunosuppression, PTA outcomes have improved significantly--except in young PTA recipients. The more potent immune system in young recipients appears to play a key role. In this study, our objective was to investigate outcomes of PTA, by recipient age, with the use of different immunosuppressive maintenance regimens. METHODS: Using information from the International Pancreas Transplant Registry and from the United Network for Organ Sharing, we analyzed outcomes of 393 technically successful enteric-drained transplants in the PTA category that were performed from January 2003 through December 2012. All PTA recipients underwent induction immunosuppression with thymoglobulin and pulse steroids and were then maintained on long-term low-dose prednisone. Excluded from our study group were patients who experienced surgical graft loss. We divided the 393 recipients into 2 age groups: <42 years (187 patients) versus ≥42 years (206 patients). For both the younger group and the older group, we compared 2 maintenance immunosuppressive regimens: (1) tacrolimus (Tac) and mycophenolate mofetil (MMF) versus (2) Tac/MMF and sirolimus (Srl). We refer to immunosuppression with Tac and MMF as the non-Srl regimen. RESULTS: The overall 3-year graft survival rate, across both age groups, was significantly better with the Srl regimen (P = .03). Regardless of the immunosuppressive regimen used, outcomes were significantly better in the older group than in the younger group (P = .05). In the older group, with both regimens, outcomes were similar (P = .55). But in the younger group, outcomes with the Srl regimen were significantly better (P = .009) than with the non-Srl regimen and, in fact, were similar to outcomes in the older group. CONCLUSIONS: Our study shows that adding Srl to the standard maintenance immunosuppressive regimen of Tac and MMF provides the best outcomes in young PTA recipients, the most immunologically robust and therefore the most immunologically challenging age group. To achieve excellent outcomes, more potent immunosuppression is required in this cohort. We think that PTA should be offered to young patients with labile diabetes before secondary complications develop.

Picadura por escorpión en Líbano. Presentación de un caso y revisión de la literatura / Scorpion sting in Lebanon. A case report

Las picaduras por escorpión son lesiones frecuentes, especialmente durante el verano, ocasionalmente graves y producidas por diferentes mecanismos. La clínica varía según la especie y la clase de veneno, e incluye efectos locales, como dolor y necrosis, y generales, entre las que encontramos manifestaciones pulmonares, cardiovasculares, neurológicas y coagulopatía, entre otras. Aún no existe un protocolo de actuación estandarizado. Las primeras medidas incluyen una evaluación inicial básica. Ante la existencia de criterios de severidad deberían colocarse dos vías de grueso calibre y deberían ser realizados exámenes complementarios como análisis de sangre, estudios radiológicos y un electrocardiograma (ECG). El tratamiento local es obligado para reducir el riesgo de infección. Presentamos a continuación un caso de una picadura doble por escorpión en la pierna derecha durante la misión «Libre Hidalgo» XIV en Líbano. El paciente abandonó el ROLE 1 24 horas después de su ingreso y permaneció asintomático durante éste (AU)

Scorpion stings are frequent injuries, especially during the summer, ocasionally severe and produced by different mechanisms. Clinical manifestations vary with species and venom type, and include local, which vary from pain to necrosis, and general effects, with pulmonary, cardiovascular and neurological manifestations and coagulopathy, among others. Standard treatment protocol is still lacking. First aid include basic initial evaluation. If there are criteria of severity two large-bore cannulae should be inserted, and complementary exams such as complete blood analysis, X-ray study and electrocardiogram should be performed. Local treatment is mandatory to reduce the risk of infection. We present a case of double scorpion bite in the right low extremity suffered during the «Libre Hidalgo» XIV mission in Lebanon. The patient was discharged from ROLE 1 24 hours after admission and remained asymptomatic during his stay (AU)

Histopathology and immunophenotype of the spleen during acute antibody-mediated rejection.

Splenectomy has been reported to have a beneficial effect in treating Acute antibody-mediated rejection (ABMR). This reason for this often rapid and profound beneficial effect is not readily apparent from what is known about normal splenic immunoarchitecture. While the spleen is rich in mature B cells, it has not been noted to be a repository for direct antibody-secreting cells. We present a case of a Native American female who received a renal transplant and developed a severe episode of ABMR. The patient was initially refractory to both plasmapheresis and IVIG. The patient underwent an emergent splenectomy with almost immediate improvement in her renal function and a rapid drop in her DR51 antibodies. Immunohistochemical stains of the spleen demonstrated abundant clusters of CD138+ plasma cells (>10% CD138 cells as opposed to 1% CD138 cells as seen in traumatic controls). Though this is a single case, these findings offer a rationale for the rapid ameliorative effect of splenectomy in cases of antibody rejection. It is possible that the spleen during times of excessive antigenic stress may rapidly turn over B cells to active antibody-secreting cells or serve as a reservoir for these cells produced at other sites.

Mycobacterial infections after kidney transplant.

We looked at mycobacterial infections occurring after a kidney transplant to determine incidence, risk factors, and outcomes. Of 3921 kidney transplants performed between 1984 and 2002, 18 (0.45%) (10 men, eight women; 11 cadaveric donor, seven living donor graft) were identified as having mycobacterial infection at some time posttransplant. Mean age at transplant was 38.3 years. Racial background was: Caucasian (n = 12), African-American (n = 2), Native Indian (n = 2), Hispanic (n = 1), and Middle Eastern (n = 1). The majority had a kidney alone (n = 14). Four recipients had simultaneous transplant of a second organ: pancreas (n = 2), islets (n = 1), and liver (n = 1). None of the 18 recipients had documented mycobacterial infection pretransplant. One recipient had a positive Mantoux test at the time of transplant and then developed pulmonary tuberculosis 4 months posttransplant; the remaining 17 patients had either negative (n = 10) or unavailable (n = 7) pretransplant Mantoux results. Mean time to infection was 3.2 years (range 1 week to 12 years). The most common site of infection was respiratory (n = 8). Other sites included musculoskeletal (n = 4), skin (n = 3), gyn (n = 1), and other (n = 2). Only three of the infections were with mycobacterial tuberculi; the others were with avium (n = 5), chelonae (n = 2), or other nontuberculous mycobacteria. Risk factors included previous TB exposure, occupational exposure, or accidental soft tissue injury. Soft tissue infections often presented as chronic unhealed wounds and required extensive surgical debridements. With mean follow-up of 12.5 years since transplant and 9.2 years since infection, 13 of the recipients are alive and well; causes of death included cardiovascular (n = 3) and sepsis (n = 2).

Expression of gp19K increases the persistence of transgene expression from an adenovirus vector in the mouse lung and liver.

Activation of the cellular immune system and subsequent lysis of vector-transduced cells by adenovirus- or transgene-specific cytotoxic T lymphocytes have been shown to limit transgene expression in animal models. The adenovirus gp19K gene product associates with major histocompatibility complex class I proteins and prevents their maturation by sequestering them in the endoplasmic reticulum. gp19K has been shown to block the ability of adenovirus-specific cytotoxic T lymphocytes to recognize virus-infected cells in vitro. To determine if gp19K expression in an adenovirus vector would increase transgene persistence, a vector that replaces the E1 region of adenovirus with an expression cassette encoding both gp19K and beta-glucuronidase was constructed. This vector produced high levels of functional gp19K in infected cells. RNase protection analysis revealed efficient expression of the gp19K gene in the mouse lung. Enhanced persistence and increased beta-glucuronidase activity were observed in the lung and liver following delivery of the gp19K-expressing adenovirus vector in B10.HTG mice but not in BALB/c mice. Since gp19K binds to both class I alleles on B10.HTG mice but only one allele on BALB/c mice, these results suggest that the major histocompatibility complex class I haplotype of mice is important in determining the effectiveness of gp19K in vivo. Since gp19K has previously been shown to interact with every human major histocompatibility complex class I allele tested, the inclusion of gp19K in gene therapy vectors may increase vector persistence in human gene therapy trials.

Transcription factor binding sites downstream of the human immunodeficiency virus type 1 transcription start site are important for virus infectivity.

When transcriptionally active, the human immunodeficiency virus (HIV) promoter contains a nucleosome-free region encompassing both the promoter/enhancer region and a large region (255 nucleotides [nt]) downstream of the transcription start site. We have previously identified new binding sites for transcription factors downstream of the transcription start site (nt 465 to 720): three AP-1 sites (I, II, and III), an AP3-like motif (AP3-L), a downstream binding factor (DBF) site, and juxtaposed Sp1 sites. Here, we show that the DBF site is an interferon-responsive factor (IRF) binding site and that the AP3-L motif binds the T-cell-specific factor NF-AT. Mutations that abolish the binding of each factor to its cognate site are introduced in an infectious HIV-1 molecular clone to study their effect on HIV-1 transcription and replication. Individual mutation of the DBF or AP3-L site as well as the double mutation AP-1(III)/AP3-L did not affect HIV-1 replication compared to that of the wild-type virus. In contrast, proviruses carrying mutations in the Sp1 sites were totally defective in terms of replication. Virus production occurred with slightly delayed kinetics for viruses containing combined mutations in the AP-1(III), AP3-L, and DBF sites and in the AP3-L and DBF-sites, whereas viruses mutated in the AP-1(I,II,III) and AP3-L sites and in the AP-1(I,II,III), AP3-L, and DBF sites exhibited a severely defective replicative phenotype. No RNA-packaging defect could be measured for any of the mutant viruses as determined by quantification of their HIV genomic RNA. Measurement of the transcriptional activity of the HIV-1 promoter after transient transfection of the HIV-1 provirus DNA or of long terminal repeat-luciferase constructs showed a positive correlation between the transcriptional and the replication defects for most mutants.

The alteration of ultrastructure and immunoreactivity of human embryonic organ of Corti tissue culture after exposure to aminoglycoside (neomycin) ototoxicity.

Using electron microscopy and GABA immunohistochemistry we evaluated the effects on human embyronic organ of Corti tissue culture of exposure to the ototoxic aminoglycoside antibiotic neomycin at a dose of 1 mM for 48 to 96 hrs. Neomycin induces the formation of multilamellar myeloid structures. These lesions, found only in the basal coil but both in inner and outer hair cells, were characteristic of the membrane-associated neomycin-induced damage. A large amount of lipofucsin and numerous lipoid vacuoles as well as vesicle-filled mound-like protrusions were also observed after exposure to neomycin. It seems there is no obvious effect on GABAergic innervation.

Value of selective second-look sonography by radiologists.

PURPOSE: To assess the usefulness of sonography performed by radiologists after a review of the sonographer's findings. MATERIALS AND METHODS: A total of 398 sonograms were obtained in 392 patients. Sonographers presented preliminary images and impressions to radiologists, who performed additional imaging and recorded their conclusions. Radiologists also attempted to predict in which cases their scan was likely to show new findings or to refute the sonographer's findings. Follow-up data were obtained whenever the sonographer's and the radiologist's findings disagreed. RESULTS: In 28 cases, the radiologist made important new findings. Positive initial findings were refuted in 24 cases. Discrepant findings were seen in 22% of cases in which additional scanning was predicted to be beneficial, compared with only 6% of cases in which second-look sonography was predicted not to be of value. This difference was statistically significant (P<.0001). CONCLUSION: Second-look sonography by radiologists provides a valuable check of the sonographer's findings.

Cyclophosphamide-induced apoptosis induces phocomelia in the mouse.

Phocomelia (absence of upper fore and/or hind limbs) was induced in mouse fetuses using cyclophosphamide. On day 11 of gestation, pregnant mice were injected intraperitoneally with 10 ml/kg of saline containing cyclophosphamide (CP) at a dosage of 20 mg/kg body weight. On day 18, the fetuses were removed by Caesarean section from dams given CP on day 11 and were examined for external anomalies. Of 22 fetuses from CP-treated dams, 13 were dead or absorbed, but the surviving 9 fetuses were found to have phocomelia with various other external anomalies. In order to examine the direct cytotoxic effect of CP on fetal limb buds, fetuses were removed at 8, 16, 24, and 48 h after CP administration on day 11, revealing the presence of frequent pyknotic nuclei and apoptotic bodies in hematoxylin and eosin (H & E) preparations. Cell-nuclei and apoptotic bodies were frequently observed by nick end-labeling in limb buds. Transmission electron microscopy demonstrated the typical changes of apoptosis. DNA extracted from the fetal limb buds submitted to CP was analysed by agarose gel electrophoresis, showing the ladder pattern characteristic of internucleosomal cleavage. These findings suggest that cyclophosphamide causes apoptosis in mouse fetal limb buds and that this process induces the external anomalies of phocomelia.

DNA fragmentation of human infarcted myocardial cells demonstrated by the nick end labeling method and DNA agarose gel electrophoresis.

Myocardial tissue taken from 19 autopsy cases of myocardial infarction were examined both by the nick and labeling method (NELM) and by DNA agarose gel electrophoresis in order to demonstrate the localization of cells with fragmented DNA and to confirm the internucleosomal cleavage of DNA biochemically. The nuclei corresponding to those with the histological features of acute myocardial infarction in hematoxylin and eosin (H&E)-stained sections were stained strongly positive with the nick end labeling method. Myocardial cells corresponding to those with nick end labeling method-stained nuclei, on the other hand, had mostly pyknotic and karyolytic nuclei and some unremarkable nuclei, even nuclear ghosts, and showed degenerated cytoplasm, including contraction band necrosis in H&E-stained preparations. The agarose gel electrophoresis of DNA extracted from the corresponding areas mentioned above showed the ladder pattern of internucleosomal cleavage characteristic of apoptosis. The present study revealed that infarcted myocardial cells with nuclear outlines, even nuclear ghosts, showed a distinct DNA fragmentation with the ladder pattern of internucleosomal cleavage. It is concluded from this study that the damaged myocardial cells of acute myocardial infarction represent a coagulation necrosis having the biochemical nature of apoptosis.

A brief study on the mixture rules of the acoustic non-linearity parameter B/A in binary biological mixtures.

Using the improved thermodynamic method, the acoustic non-linearity parameter B/A of binary biological mixtures--such as pig blood, sheep blood and bovine blood--is measured in order to test the mixture rules proposed by Apfel and Sehgal. The results show that the B/A values of the above-mentioned blood decrease with an increasing volume fraction of water. The B/A value varies linearly with the volume fraction of protein. It is also shown that, treated as a binary mixture, the blood obeys the trend that the B/A value varies with the volume of water. This is in agreement with the rules proposed by Apfel and Sehgal. However, the measured B/A value is not exactly equal to that of the mixture rules.

SEM observation and GABA immunoreactivity of human embryonic organ of Corti tissue culture.

Scanning electronic microscopy (SEM) observation and GABA immunoreactivity of the tissue culture of 6 human embryonic organs of Corti are reported. The normal structural appearance of the hair cells and the facts that there were no bulging of cuticular plates, and cytoplasmic protrusion proved that the hair cells were healthy. After GABA immunostaining, radial bundles, innerspiral bundles, and the ending of inner and outer hair cell regions displayed typical GABA-positive reactivity. The results indicate that the human embryonic organ of Corti is well developed differentiated, and mature at 19 gestational weeks and can continue to grow and differentiate in vitro. The study offers an ideal and important biological model for further research into the human ear.