Acute kidney injury caused by tenofovir disoproxil fumarate and diclofenac co-administration.

OBJECTIVES: The renal elimination of tenofovir (TFV) may be subject to renal drug-drug interactions that may increase the risk of kidney injury. Case reports indicated that diclofenac might increase TFV-associated nephrotoxicity via a drug-drug interaction, leading to an increased intracellular TFV concentration in proximal tubular cells. METHODS: A retrospective analysis of data for all patients from the Frankfurt HIV Cohort (FHC) who had diclofenac prescriptions between January 2008 and June 2012 was carried out. RESULTS: Among 89 patients with diclofenac use, 61 patients (68.5%) were treated with tenofovir disoproxil fumarate (TDF) and 28 patients (31.5%) were treated with TDF-sparing combination antiretroviral therapy (cART). Thirteen patients (14.6%) developed acute kidney injury (AKI) shortly after initiating diclofenac treatment. AKI occurred exclusively in TDF-treated patients, although all had previously stable renal function. All cases were accompanied by new onset of at least two parameters indicating proximal tubular damage, such as normoglycaemic-glucosuria and hypophosphataemia. TFV-associated nephrotoxicity was demonstrated by renal biopsy in four cases. Additionally, 11.5% of patients on TDF treatment developed new-onset proximal tubular damage, while having a preserved glomerular filtration rate. In contrast, diclofenac did not affect renal function in patients with TDF-sparing cART, as only one case of isolated hypophataemia was observed in these patients. In univariate analysis, risk factors for AKI were TDF-containing cART (P = 0.0076) and pre-existing hypophosphataemia (P = 0.0086). CONCLUSIONS: Drug-drug interaction caused by diclofenac could exacerbate TFV-associated nephrotoxicity. Diclofenac should be used with caution in patients on TDF therapy, especially in those with hypophosphataemia. Our findings need to be confirmed in larger studies.

Skin tolerance is supported by the spleen.

The repeated application of antigens results in the induction of tolerance. Lymph nodes are responsible for this reaction by producing suppressor cells. Using an in vivo transplantation model, we showed recently that stromal cells from different lymph nodes induce different cell populations for suppression, which all produce a tolerogenic phenotype. In this study, we were interested in the role of the spleen in these tolerance reactions. Therefore, tolerance was induced via feeding or injecting ovalbumin several times in control and splenectomized mice. The delayed-type hypersensitivity (DTH) was measured as well as the cell subset composition of the spleen. The spleen of peripherally tolerized mice showed higher proliferation activity and a specific antibody production compared with orally tolerized mice, where regulatory T cells were predominantly found. Tolerance induction after removal of the spleen resulted in a reduced DTH response in antigen fed animals, whereas skin tolerance induction failed. In conclusion, the results illustrate that lymph nodes from different areas employ their individual pathways for similar immune reactions, and the spleen is part of this reaction initiated at the peripheral site.

c-Myc and EBV-LMP1: two opposing regulators of the HLA class I antigen presentation machinery in epithelial cells.

BACKGROUND: Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) up-regulates the human leukocyte antigen (HLA) class I antigen presentation machinery (APM). This appears counterintuitive with immune evasion in EBV-associated tumours like nasopharyngeal carcinoma (NPC). METHODS: Latent membrane protein 1-transfected epithelial cell lines were used as a model system to study the impact of LMP1 and c-Myc on HLA class I components. The expression of components of the HLA class I APM, c-Myc and Ki-67 was analysed in LMP1+ and LMP1- NPC by immunohistochemistry. RESULTS: In epithelial cells, LMP1 up-regulated HLA class I APM. This effect could be counteracted by c-Myc, which itself was up-regulated by LMP1 apparently through IL6 induction and Jak3/STAT3 activation. Studies of NPC biopsies revealed down-regulation of HLA class I APM expression. No difference was observed between LMP1+ and LMP1- NPC. However, expression of Ki-67 and c-Myc were up-regulated in LMP1+ tumours. CONCLUSION: These findings raise the possibility that c-Myc activation in NPC might antagonise the effect of LMP1 on HLA class I expression thus contributing to immune escape of tumour cells.

Lymph node dissection--understanding the immunological function of lymph nodes.

Lymph nodes (LN) are one of the important sites in the body where immune responses to pathogenic antigens are initiated. This immunological function induced by cells within the LN is an extensive area of research. To clarify the general function of LN, to identify cell populations within the lymphatic system and to describe the regeneration of the lymph vessels, the experimental surgical technique of LN dissection has been established in various animal models. In this review different research areas in which LN dissection is used as an experimental tool will be highlighted. These include regeneration studies, immunological analysis and studies with clinical questions. LN were dissected in order to analyse the different cell subsets of the incoming lymph in detail. Furthermore, LN were identified as the place where the induction of an antigen-specific response occurs and, more significantly, where this immune response is regulated. During bacterial infection LN, as a filter of the lymph system, play a life-saving role. In addition, LN are essential for the induction of tolerance against harmless antigens, because tolerance could not be induced in LN-resected animals. Thus, the technique of LN dissection is an excellent and simple method to identify the important role of LN in immune responses, tolerance and infection.

Influence of systolic-pressure-variation-guided intraoperative fluid management on organ function and oxygen transport.

BACKGROUND: Dynamic variables, for example, systolic pressure variation (SPV), are superior to filling pressures for assessing fluid responsiveness. We analysed the effects of SPV-guided intraoperative fluid management on organ function and perfusion when compared with routine care. METHODS: Eighty patients (44 female and 36 male) undergoing elective major abdominal surgery were randomly assigned to a control group [n=40, mean age 66 (sd 10), range 40-84 yr] or SPV group [n=40, age 61 (16), range 26-100 yr] in which intraoperative fluid management was guided by SPV (trigger: SPV>10%). Central venous O2 saturation (ScvO2), lactate and bilirubin, creatinine, indocyanine green plasma disappearance rate (ICG-PDR), and gastric mucosal CO(2) tension were measured after induction of anaesthesia, after 3, 6, 12, and 24 h. RESULTS: Patient characteristics, duration of surgery [5.8 (2.5) vs 5.4 (2.5) h], and infusion volumes (median 4865 vs 4330 ml) were comparable between the groups. At 3 and 6 h, SPV (P=0.04, P=0.01) and Deltadown (P=0.005, P=0.01) were significantly higher in the control group. Oxygen transport and organ function were comparable: baseline and 24 h values for ICG-PDR: 28.5 (7.9) and 22.7 (7.8) vs 23.9 (6.9) and 26.1 (5.9)% min(-1), 77.7 (6.6) and 72.6 (5.5) vs 79.3 (7.1) and 72.8 (6.7)% for ScvO2 and 1.0 (0.4) and 1.2 (0.6) vs 0.9 (0.2) and 1.3 (0.5) mmol litre(-1) for lactate. Length of mechanical ventilation, ICU stay, and mortality were comparable. CONCLUSIONS: In comparison with routine care, intraoperative SPV-guided treatment was associated with slightly increased fluid adminstration whereas organ perfusion and function was similar.

Investigation of intra-herd spread of Mycobacterium caprae in cattle by generation and use of a whole-genome sequence.

Single nucleotide polymorphisms (SNPs) calculated from whole genome sequencing (WGS) are ideally suited to study evolutionary relationships of pathogens and their epidemiology. Mycobacterium caprae infections have been documented frequently in cattle and red deer along the Bavarian and Austrian Alps during the last decade. However, little is still known about the transmission within cattle holdings and possible alterations of the genomes of M. caprae during such events. The aim of this study was to study the molecular epidemiology of bovine tuberculosis (bTB) in selected herds based on isolate-specific genome-wide SNPs and to perform a phylogenetic network analysis. In total, 61 M. caprae isolates were collected originating from eight cattle farms over a period of twelve years between 2004 and 2015. Analysis of their sequence data revealed that the M. caprae isolates of an affected farm differ at all in a few SNPs. In contrast, many more SNPs were found when comparing the M. caprae genomes originating from different herds. The results demonstrated that the spread of bTB in the affected farms occurred by direct transmission between the members of each herd rather than between herds and a M. caprae introduction in farms after contact events e. g. on summer pastures can readily be traced by WGS analysis. Furthermore, we assembled a nearly complete whole genome sequence of M. caprae derived from several cattle isolates originating from bTB cases in the Bavarian Alpine region.

The Sheep Grimace Scale as an indicator of post-operative distress and pain in laboratory sheep.

The EU Directive 2010/63/EU changed the requirements regarding the use of laboratory animals and raised important issues related to assessing the severity of all procedures undertaken on laboratory animals. However, quantifiable parameters to assess severity are rare, and improved assessment strategies need to be developed. Hence, a Sheep Grimace Scale (SGS) was herein established by observing and interpreting sheep facial expressions as a consequence of pain and distress following unilateral tibia osteotomy. The animals were clinically investigated and scored five days before surgery and at 1, 3, 7, 10, 14 and 17 days afterwards. Additionally, cortisol levels in the saliva of the sheep were determined at the respective time points. For the SGS, video recording was performed, and pictures of the sheep were randomized and scored by blinded observers. Osteotomy in sheep resulted in an increased clinical severity score from days 1 to 17 post-surgery and elevated salivary cortisol levels one day post-surgery. An analysis of facial expressions revealed a significantly increased SGS on the day of surgery until day 3 post-surgery; this elevated level was sustained until day 17. Clinical severity and SGS scores correlated positively with a Pearson´s correlation coefficient of 0.47. Further investigations regarding the applicability of the SGS revealed a high inter-observer reliability with an intraclass correlation coefficient of 0.92 and an accuracy of 68.2%. In conclusion, the SGS represents a valuable approach for severity assessment that may help support and refine a widely used welfare assessment for sheep during experimental procedures, thereby meeting legislation requirements and minimizing the occurrence of unrecognized distress in animal experimentation.

Stromal cells as trend-setters for cells migrating into the lymph node.

Lymph node stromal cells are known to be immunorelevant during inflammation and tolerance. Differences between peripheral lymph nodes and mesenteric lymph nodes are important for an efficient and effective immune defense. Stromal cells were considered to be perfectly adapted to their draining area and not changeable concerning their expression pattern. Here we show that stromal cells can change their profile after isolation and transplantation into a different draining area. Subsequently, these newly organized lymph nodes are able to induce not only a region-specific but also an antigen-specific immune response. Thus, stromal cells are trend-setters for immune cells in producing a microenvironment that allows an optimized immune defense.

Serological and antigenical findings indicating pestivirus in man.

An epidemiological survey for pestivirus was undertaken in Zambia and Europe, in view of the recent serological findings obtained by previous studies in Europe with humans. Collected sera were tested for anti-bovine viral diarrhea virus (BVDV) specific antibodies by IIF and Western Blotting. Of those individuals tested (n = 1272), 15.3% showed a seropositive reaction to the BVDV. Anti-BVDV antibody prevalence in immuno-depressed patients (e.g. HIV positive) was investigated. A higher prevalence was revealed in HIV patients suffering from chronic diarrhoea and in those having developed AIDS Related Complex (ARC). Our of 212 persons tested for pestivirus isolation, a non cytopathic virus strain was detected in 2 buffy coat samples using IIF with a specific anti-BVDV serum. The isolation could be repeated three times during 31 days in one person. The virus was identified as a pestivirus with radioimmuno-precipitation assays and IIF-flow cytometry. A doublet of 120 kD was identified only in cell lysates, indicating a non-structural protein. In order to rule out cross reactivity 30 sera from Hepatitis C seropositive patients were tested against the isolate by IIF-flow cytometry. No antigen-specific binding could be observed. These findings indicated the occurrence of a pestivirus in man and might suggest a relationship with a pestivirus of animal origin.

Falls in the rehabilitation setting: incidence and characteristics.

A prospective six-month study was conducted to determine a high-risk index for medical rehabilitation patients who fall. Variables studied for all patients included demographics, medical conditions, associated symptoms, orthostatic blood pressure measurements, physical function, posture control, proprioception, use of physical restraints, and medications, A detailed examination of the fall events was also conducted. Of the 143 patients studied, 46 (32%) fell at least once, making a total of 84 falls. Impaired ability to follow directions, impaired judgment, impaired proprioception, presence of physical restraints, use of major tranquilizers, use of sedatives, and presence of psychiatric diagnosis were all individually associated with patients who fell. Males fell more than females. Logistic regression identified altered proprioception as the only major predictor of falling. Of those who fell, only 26% called for assistance prior to the fall. Sixty-eight percent of the falls were from wheelchairs. Importantly, no patients had serious injury or morbidity from the falls.

Frequently selected nursing diagnoses for the rehabilitation client with stroke.

From 2 million to 3 million people in the United States live with the aftereffects of stroke. Nursing diagnoses provide a taxonomy that enables nurses to identify similarities and differences for given groups of clients. The purposes of this study were to identify the most frequently chosen nursing diagnoses for rehabilitation stroke clients and to determine the corresponding objective clinical characteristics (related factors) of these diagnoses. A retrospective descriptive design was used to study charts from randomly selected stroke clients (N = 100) at a large rehabilitation center. At admission and at discharge, impaired physical mobility (99%) and self-care deficit (91%) were the most frequently occurring diagnoses. Impaired physical mobility was usually related to neuromuscular impairment, and self-care deficit was usually related to neuromuscular dysfunction. These objective clinical characteristics help to determine how diagnoses are unique to rehabilitation nursing practice.

The intestinal micro-environment imprints stromal cells to promote efficient Treg induction in gut-draining lymph nodes.

De novo induction of Foxp3⁺ regulatory T cells (Tregs) is particularly efficient in gut-draining mesenteric and celiac lymph nodes (mLN and celLN). Here we used LN transplantations to dissect the contribution of stromal cells and environmental factors to the high Treg-inducing capacity of these LN. After transplantation into the popliteal fossa, mLN and celLN retained their high Treg-inducing capacity, whereas transplantation of skin-draining LN into the gut mesenteries did not enable efficient Treg induction. However, de novo Treg induction was abolished in the absence of dendritic cells (DC), indicating that this process depends on synergistic contributions of stromal and DC. Stromal cells themselves were influenced by environmental signals as mLN grafts taken from germ-free donors and celLN grafts taken from vitamin A-deficient donors did not show any superior Treg-inducing capacity. Collectively, our observations reveal a hitherto unrecognized role of LN stromal cells for the de novo induction of Foxp3⁺ Tregs.

Activation-induced cytidine deaminase (AID) is expressed in normal spermatogenesis but only infrequently in testicular germ cell tumours.

Activation-induced cytidine deaminase (AID) is essential for somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin genes in antigen-dependent B-cell maturation. SHM is not restricted to immunoglobulin gene loci, raising the possibility of a function for AID in other cell types. In this study, it is shown that AID is expressed in spermatocytes in the human testis. AID was mostly cytoplasmic but nuclear AID was also observed in a proportion of cells, in keeping with the DNA deamination model of AID function. Intratubular germ cell neoplasia unclassified (IGCNU), the precursor lesion of testicular cancers, was AID-negative. Seminomas also lacked AID expression. Nuclear and cytoplasmic AID expression was observed in three of 32 mixed non-seminomatous germ cell tumours. The results provide evidence for a physiological role for AID outside the immune system. AID expression in spermatocytes points to a role in meiosis. It remains uncertain whether AID may also contribute to the genetic aberrations characteristically found in testicular germ cell tumours. The consistent absence of detectable AID expression in atypical spermatogonia of IGCNU and its rare expression in germ cell tumours suggest that continued expression of AID is not involved in the pathogenesis of germ cell tumours.

The antibody 2B4 directed against the Epstein-Barr virus (EBV)-encoded nuclear antigen 1 (EBNA1) detects MAGE-4: implications for studies on the EBV association of human cancers.

We have previously developed two monoclonal antibodies against the Epstein-Barr Virus (EBV) nuclear antigen 1 (EBNA1), designated 1H4 and 2B4. Both detect EBNA1 by in situ staining in established EBV-positive tumours, e.g. Hodgkin's lymphoma and nasopharyngeal carcinoma. An association of EBV with other tumours, notably breast carcinomas, has been reported but remains controversial. Using the antibody 2B4, a nuclear protein has been detected in breast carcinomas that were EBV-negative by other methods, suggesting cross-reactivity with a cellular protein. Furthermore, an association of EBV with various other carcinomas has been reported on the basis of 2B4 immunohistochemistry. Here we show that 2B4 also binds to MAGE-4, a cancer testis antigen expressed in a variety of tumour cells, including breast carcinoma, seminoma and EBV-negative cases of Hodgkin's lymphoma. We conclude that the 2B4 antibody is not suitable for the detection of EBV infection but that additional techniques, particularly in situ hybridization for the detection of the EBV-encoded RNAs (EBERs), should be employed to confirm the presence of EBV. Our results add to the evidence indicating that breast cancer is not an EBV-associated disease.

Cyclic adenosine 3',5'-monophosphate in Escherichia coli.

The concentration of cyclic adenosine 3',5'-monophosphate (c-AMP) in Escherichia coli growing on different sources of carbon was studied. Cultures utilizing a source of carbon that supported growth relatively poorly had consistently higher concentrations of c-AMP than did cultures utilizing sugars that supported rapid growth. This relationship was also observed in strains defective in c-AMP phosphodiesterase and simultaneously resistant to catabolite repression; in such strains the c-AMP concentration was slightly higher for several sources of carbon tested. Cultures continued to synthesize c-AMP and secreted it into the medium, under conditions that brought about an inhibition of the intracellular accumulation of the cyclic nucleotide. Transient repression of the synthesis of beta-galactosidase was not associated with an abrupt decrease in the cellular concentration of c-AMP.

Cystathionine metabolism in methionine auxotrophic and wild-type strains of Saccharomyces cerevisiae.

The role of cystathionine in methionine biosynthesis in wild-type and auxotrophic strains of Saccharomyces cerevisiae was studied. Homocysteine and cysteinerequiring mutants were selected for detailed study. Exogenously supplied cystathionine, although actively transported by all strains tested, could not satisfy the organic sulfur requirements of the mutants. Cell-free extracts of the wild-type, homocysteine, and cysteine auxotrophs were shown to cleave cystathionine. Pyruvic acid and homocysteine were identified as teh products of this cleavage. A mutant containing an enzyme which could cleave cystathionine to homocysteine in cell-free experiments was unable to use cystathionine as a methionine precursor in the intact organisms. The significance of this finding is discussed.

Immune phenotype of canine hematopoietic progenitor cells.

The immune phenotype of canine hematopoietic progenitor cells was studied by immunoseparation and culturing of separated cells. Two separation methods were used, the magnetic cell sorting system (MACS) and the fluorescence activated cell sorter (FACS). For separation rat anti dog antibodies Dog 13 and Dog 14 directed against Thy-1, and Dog 26 as well as cross-reactive mouse anti human antibodies IOT2a and 7.2 directed against MHC class II were used. Separated cell populations were cultured in semisolid agar before and after long-term culture on a pre-established irradiated stromal cell layer. After 28 days, adherent and nonadherent cells were harvested from long-term culture. The MACS system allowed separation of cells into positive and negative fractions. Long-term culture-initiating cells (LTC-IC) were found in both the Thy-1+ and the Thy-1- fraction, but the content of LTC-IC was higher in the Thy-1+ fraction. The MACS system did not allow separation of progenitor cells according to the expression of MHC class II antigen detected by Dog 26 and the cross-reactive antibodies IOT2a and 7.2. In contrast to the MACS system the FACS allowed separation of negative, low-positive and high-positive cell populations. Low-positive fractions were well defined for Thy-1 and less well defined for MHC class II. CFU before and after long-term culture were exclusively observed in the low positive fraction (Thy-1(lo+)). Using MHC class II antibody Dog 26 LTC-IC were found mainly in the negative and low positive fraction, and CFU were observed mainly in the low and high positive fraction. In conclusion pluripotent canine hematopoietic precursor cells are low positive for Thy-1 and for MHC class II. In this respect canine hematopoietic progenitor cells are comparable to those of mouse and man.