Role of Gag mutations in PI resistance in the Swiss HIV cohort study: bystanders or contributors?

Background: HIV Gag mutations have been reported to confer PI drug resistance. However, clinical implications are still controversial and most current genotyping algorithms consider solely the protease gene for assessing PI resistance. Objectives: Our goal was to describe for HIV infections in Switzerland the potential role of the C-terminus of Gag (NC-p6) in PI resistance. We aimed to characterize resistance-relevant mutational patterns in Gag and protease and their possible interactions. Methods: Resistance information on plasma samples from 2004-12 was collected for patients treated by two diagnostic centres of the Swiss HIV Cohort Study. Sequence information on protease and the C-terminal Gag region was paired with the corresponding patient treatment history. The prevalence of Gag and protease mutations was analysed for PI treatment-experienced patients versus PI treatment-naive patients. In addition, we modelled multiple paths of an assumed ordered accumulation of genetic changes using random tree mixture models. Results: More than half of all PI treatment-experienced patients in our sample set carried HIV variants with at least one of the known Gag mutations, and 17.9% (66/369) carried at least one Gag mutation for which a phenotypic proof of PI resistance by in vitro mutagenesis has been reported. We were able to identify several novel Gag mutations that are associated with PI exposure and therapy failure. Conclusions: Our analysis confirmed the association of Gag mutations, well known and new, with PI exposure. This could have clinical implications, since the level of potential PI drug resistance might be underestimated.

Treatment of fulminant acute Hepatitis B with nucles(t)id analogues is safe and does not lead to secondary chronification of Hepatitis B.

Background: Acute hepatitis B virus (HBV) infection is still a major cause of acute liver failure (ALF), necessitating a high rate of emergency liver transplantation (LTx). Acute infection is followed by high viral replication rates leading to hepatocyte death and, ultimately, ALF. The objective of treating HBV-induced ALF thus is to eliminate, or significantly suppress, HBV replication and therefore reduce cell death and support regeneration. Objective: In this retrospective study, we want to evaluate the timing, the safety, and the long-term virological outcome of this approach. Methods/results: In this study, we included 32 patients (16 female and 16 males; median age 39.5 years) with ALF due to hepatitis B, who were transferred to the university hospital Essen, Germany between January 2009 and December 2013. Before treatment, transaminases were highly elevated, bilirubin was increased, and elevated international normalized ratio (INR) revealed impaired liver function. HBV-DNA and HBsAg were positive. All 32 patients received oral antiviral treatment (3 lamivudine, 21 entecavir, and 8 tenofovir) between 1 day and 4 months after diagnosis of acute hepatitis B. One patient died, 2 were transplanted, one died shortly after LTx the other patient survived after LTx. These 3 patients received treatment in a state of advanced liver failure, and 1 patient 4 months after initial diagnosis of hepatitis B. Twenty-nine patients survived without LTx. Five patients were discharged without further follow-up. All 24 remaining patients became HBV-DNA negative in median of 100 days. Twenty-two patients were followed further, and all patients lost their HBsAg in median of 108 days. Sixteen of the 22 patients experienced a seroconversion to anti-HBs in median of 137 days. Four patients who were followed for 1 more year after HBsAg did not develop anti-HBs. None of the patients developed chronic hepatitis B. Conclusion: Immediate treatment of HBV-induced ALF with nucleos(t)id-analogues (NUCs) appears save and prevents LTx and death, and there is no indication for increased chronicity.

Early identification of renal transplant recipients with high risk of polyomavirus-associated nephropathy.

Polyomavirus BK (BKPyV) is ubiquitous among humans. Following primary infection, the virus remains latent predominantly in the hosts' uroepithelial cells. Up to 10 % of renal transplant recipients show a viral reactivation that can lead to polyomavirus-associated nephropathy (PyVAN). In the absence of early treatments, the risk of graft loss is up to 80 %. Monitoring viral load in urine and plasma by real-time PCR after transplantation is the most common diagnostic tool to detect viral reactivation. In the present retrospective study, BKPyV-DNA loads in urine and plasma by quantitative real-time PCR were associated with clinical data, including HLA haplotype, blood parameters and viral genotype, of 40 renal transplant recipients at the University Clinics of Cologne. Seventeen out of 329 patients screened for BKPyV from January 2009 to October 2013 were detected BKPyV positive in urine only, whereas in 23 patients the virus became additionally detectable in plasma. Among these, ten patients progressed to PyVAN. Overall, the present study showed that the detection from the third month onwards after transplantation of a first viruric episode with a median viral load of 1 × 10(8) copies/mL, followed after few days by a first viremic episode with a median viral load of >1 × 10(4) copies/mL, was strongly associated with the development of PyVAN. In conclusion, the viral load and the temporal profile of the first viruric and viremic episode post-transplantation, in combination with specific features of the host immune response, should be considered as relevant clinical determinants of the risk of renal transplant recipients to progress to PyVAN.

Pandemic 2009 influenza A(H1N1) virus infection coinciding with invasive pulmonary aspergillosis in neutropenic patients.

SIn patients receiving anti-neoplastic chemotherapy, the impact of influenza on the incidence of invasive pulmonary aspergillosis (IPA) remains unknown. We matched data of the Cologne Cohort of Neutropenic Patients (CoCoNut) with records from the Institute for Virology and compared the findings to historical data. During the pandemic, we diagnosed influenza A(H1N1) in five patients with malignancies and febrile neutropenia refractory to antibiotic therapy. Probable IPA was diagnosed in three of these patients on the grounds of typical computed tomography morphology and microbiological results. Three of five patients receiving remission-induction chemotherapy for acute myeloid leukaemia developed aspergillosis although receiving posaconazole prophylaxis. In the 3 years before the influenza pandemic, only 2/77 patients of this group developed infection. Infection with influenza A(H1N1) may increase the risk for invasive aspergillosis in neutropenic patients. Pulmonary aspergillosis is an important additional differential diagnosis in neutropenic influenza patients with pneumonia.

Transmission of human immunodeficiency virus I drug resistance - a case report. What are the clinical implications?

The success of first-line antiretroviral therapy can be challenged by the acquisition of primary drug resistance. Here we report a case where baseline genotypic resistance testing detected resistance conferring nucleoside/nucleotide reverse transcriptase inhibitor (NRTI)-associated mutations, but no primary mutations for protease inhibitor (PI). Subsequent PI-based HAART with boosted saquinavir led to virological treatment success with persistently undetectable viral load. After treatment simplification from saquinavir to an atazanavir based PI-therapy and no change in backbone therapy rapid virological breakthrough occurred. Retrospective analysis displayed preexisting gag cleavage site mutations which may have reduced the genetic barrier in a clinical relevant manner in combination with the already existing NRTI resistance mutations. Alternatively, this effect could be explained with a different antiviral potency for the respective PIs used.

[Fever of unknown origin caused by anaplasmosis]. / Koorts van onbekende oorsprong, met als oorzaak anaplasmose.

A 74-year-old man in good clinical condition presented with complaints of recurrent fever up to 38 degrees C and diffuse thoracic pain, both present for several weeks. Antibiotic therapy did not result in disappearance of the symptoms. Except for a picture of chronic inflammation and a positive Anaplasma IgM antibody titre, serology yielded no indication ofthe diagnosis. Further investigation, including imaging, showed no convincing cause. 'Anaplasma infection' remained the working diagnosis. One month after hospitalisation, the patient was free of fever without any type of therapy. Control blood tests revealed seroconversion to Anaplasma IgG antibodies, constituting serological evidence of a recent infection. The prevalence ofanaplasmosis is increasing. Mice are the principal reservoir for the intracellular bacteria and the infection is transmitted by ticks. In case of fever of unknown origin, since the transmission takes place in the same manner, one should consider both Borrelia and an infection with Anaplasma.

Co-operation of IL-1 and IL-2 on T-cell activation in mononuclear cell cultures.

In search of an optimized anti-cancer immunotherapy, the combination of IL-2 and IL-1 has been tried. In an in-vitro LAK model, this cytokine cocktail seemed to be quite promising. In our in-vitro model of IL-2 induced T-cell activation we have therefore investigated the co-operation of these two potent immunostimulators. Mononuclear cells were stimulated with CD3 activating antibody in the presence of different cytokines and blocking or neutralizing antibodies. Cytokine concentrations were detected in the supernatants with ELISA. Intracellular IFN-gamma and IL-4 in the different T-cell subsets was measured by flow cytometry. IL-1 and IL-1 receptor antagonist (IL-1Ra) were up-regulated by IL-2, this was achieved independently of IL-12 or CD40/CD40L interaction. As a negative feedback mechanism, IL-1beta induced its natural antagonist, IL-1Ra. Both endogenous and exogenous IL-10 suppressed IL-1beta and induced IL-1Ra, thus markedly decreased the amount of functional IL-1. The combination of IL-2 and IL-1beta lead to a mildly increased Interferon-gamma (IFN-gamma) secretion (+20%, p < 0.05), however, this appeared to be the result of an increased IFN-gamma production per secreting cell, rather than of an increased recruitment of non-secreting cells. Similarly, IL-6 was also induced in an additive fashion (+30%, p < 0.05). For both cytokines, this effect could be significantly augmented by neutralizing IL-1Ra. Concentrations of IL-2 induced IL-10 and soluble Fas ligand (sFasL) were not affected by IL-1beta. We were thus able to demonstrate that IL-1 relays its activity through different pathways than IL-2. Furthermore, we could show that the potentially synergistic action of IL-2 and IL-1 was hindered by the simultaneous induction of signficant amounts of IL-1Ra. From the latter findings we conclude that the combination of IL-2 and IL-1 for cytokine-induced anti-tumor activity may not, but a combination of IL-2 and anti-IL-1Ra might prove beneficial.

[Follow-up of patients with osteosarcoma and Ewing's sarcoma: a retrospective cost-benefit analysis]. / Nachsorge bei Patienten mit Osteosarkom und Ewing-Tumoren. Eine retrospektive Nutzenanalyse.

PURPOSE: Determination of the respective roles of clinical investigation, laboratory tests and various imaging techniques in the follow up of children and adolescents with osteosarcoma and Ewing's sarcoma. METHODS: In a retrospective monocenter analysis, charts of 72 patients with osteosarcoma and 47 patients with Ewing's sarcoma were reviewed with respect to ability of different diagnostic methods to detect the relapse, and correlated outcome. RESULTS: In about 25% of relapses, a second remission could be achieved. The most sensitive methods to detect a potentially curable relapse were clinical investigations and chest x-ray in the case of osteosarcoma and chest x-ray and whole body scintigraphy in the case of Ewing's sarcoma. CONCLUSIONS: The different value of diagnostic methods in the follow-up of the two illnesses may be explained by the different tumor biologies and by distinct therapeutic strategies for the treatment of relapses in the two tumor entities. However, an ongoing evaluation of current follow-up strategies is necessary to take into account new therapeutic developments which may shift the importance of certain imaging techniques.

In vivo cytokine responses to interleukin-2 immunotherapy after autologous stem cell transplantation in children with solid tumors.

The potent immunostimulatory cytokine interleukin-2 (IL-2) has been extensively investigated for its potential to induce anti-tumor immunity in a number of tumor models. Only recently the complex interplay of mutually suppressive or supportive cytokines of the IL-2-induced network of cytokines has been better characterized. The aim of this study was to assess which of these in vitro findings are reproducible in vivo in recipients of stem cell transplants (SCT), since in these patients long- lasting impairments in cytokine inducibility have been described. We have therefore studied the kinetics of putative modulators and mediators of IL-2-induced immune activation, namely IL-1beta, IL-4, IL-5, IL-10, IL-12, soluble Fas ligand (sFasL), and GM-CSF during IL-2 therapy. All patients were children or adolescents suffering from solid tumors with poor prognosis who received three 5-day courses of high-dose intravenous IL-2 as an adjuvant to their radio-chemotherapy and autologous SCT. While IL-1beta, IL-4 and IL-12 were not, and sFasL was only mildly affected by the IL-2 therapy, we observed a consistent and early rise of IL-10, IL-5, and GM-CSF. These increases were rapidly reversible after discontinuation of IL-2 therapy. The inducibility of IL-10, IL-5 and GM-CSF was more pronounced with increasing time from the SCT, and in the third cycle reached an order of magnitude as in high-dose IL-2 patients without SCT. Together with the abundant in vitro data, these findings may help devise a combination immunotherapy permitting stronger anti-tumor effects, but lesser adverse effects.

Regulation of interleukin-2 induced interleukin-5 and interleukin-13 production in human peripheral blood mononuclear cells.

Adjuvant interleukin (IL)-2 immunotherapy has been used for many years for a variety of malignant and nonmalignant entities. In many cases, a dose escalation might have seemed desirable, but was prevented by the rather severe adverse effects of systemic IL-2 application. Only recently has the regulation of IL-2 induced cytotoxicity been understood better, so that now efforts can be aimed at the design of cytokine cocktails that would selectively induce cytotoxicity but result in as few adverse effects as possible. Previously, induction of IL-5 under systemic IL-2 therapy has been described, and a number of the side-effects have been attributed to this event. We therefore investigated the regulation of IL-2 induced production of IL-5 and IL-13 (which, similarly to IL-5, is a mediator of allergy-like symptoms). At the same time, the effects of regulatory cytokines, such as IL-4, IL-10 and IL-12, on interferon-gamma (IFN-gamma), the major cytotoxic mediator of IL-2 therapy, were studied. All three have been discussed as antitumour immunotherapeutics, either alone or in combination with IL-2. In anti-CD3-treated peripheral blood mononuclear cells, IL-2 induced IL-5 and IL-13 alongside IFN-gamma, IL-10 and IL-12. In the presence of IL-2, inhibition of endogenous IL-12 production further enhanced the IL-5 and IL-13 responses, while IFN-gamma and IL-10 were markedly suppressed. Co-incubation with IL-2 and IL-12 suppressed IL-5/IL-13 below, but enhanced IFN-gamma and IL-10 above, levels induced by IL-2 alone. IL-10 was suppressive on all the investigated cytokines, while IL-4 interfered with IL-2 induced IFN-gamma and IL-12 production, but was additive to IL-2 in its effect on IL-5 and IL-13. These data suggest that the combination of IL-12 with IL-2 would enhance the cytotoxic activity of this regimen, but might reduce its adverse effects.

Transforming growth factor-beta1 suppresses interleukin-15-mediated interferon-gamma production in human T lymphocytes.

One of the most remarkable means by which tumour cells manage to evade recognition and elimination by the immune system is the release of immunosuppressive mediators, such as interleukin (IL)-10 or transforming growth factor-beta (TGF-beta). For antitumour immunotherapies to reach their full potential, cytokine cocktails will have to be custom-tailored to the tumour's individual cytokine microenvironment. One of the components of such a cytokine cocktail may be interleukin (IL)-15, which has demonstrated an excellent stimulatory potential of antitumour immunity. In an in vitro model, we have previously been able to show that the negative effects of IL-10 on IL-15-mediated cytotoxic T-cell activation can be outweighed by the addition of interleukin (IL)-12. The mechanism by which TGF-beta may influence the effect of IL-15 remains poorly understood, however. We have therefore taken our T-cell model further and have studied the effect of TGF-beta on IL-15-mediated interferon-gamma (IFN-gamma) production. In activated, IL-15-stimulated peripheral blood T lymphocytes, TGF-beta suppressed IFN-gamma mRNA and protein levels by approximately 75%. This effect was likewise observed on both CD4+ and CD8+ T cells and, in contrast to the effect of IL-10 in this system, could not be neutralized by the addition of IL-12. Thus, immunotherapy for TGF-beta-producing tumours may benefit from the addition of TGF-neutralizing activity rather than IL-12.

Interleukin 10 inhibits TNF-alpha production in human monocytes independently of interleukin 12 and interleukin 1 beta.

Previously we demonstrated that endogenously produced Interleukin (IL-)10 suppressed the production of tumor necrosis factor-alpha (TNF-alpha) in CD3 activated T-cells via down-regulation of paracrine IL-12 secretion from APC. Here we investigated the effect of endogenous IL-10 on TNF-alpha production in purified lipopolysaccharide (LPS) stimulated monocytes and its mechanism. Similarly to its effects on T-cells, IL-10 inhibited monocyte TNF-alpha production by about half. Unlike in T-cells, however, this effect was not mediated via IL-12. While blockade of endogenous IL-10 binding to the IL-10 receptor enhanced the autocrine production of TNF-alpha, IL-12 and IL-1 beta, the neutralization of IL-12 or IL-1 beta did not affect the IL-10 effects on TNF-alpha production. This suggests that despite its inhibitory effects on IL-12 and IL-1 beta, which is quite similarly observed in T-cells, in purified monocytes IL-10 does not effect its TNF-alpha suppression by this mechanism. These findings indicate that IL-10 regulates production of pro-inflammatory cytokines by distinct mechanisms in different cells and tissues. Our study thus adds to the appreciation of the complex cytokine regulation of the immune system.

Evaluation of follow-up investigations in osteosarcoma patients: suggestions for an effective follow-up program.

BACKGROUND AND PROCEDURE: Follow-up programs for cancer patients aim at improving the overall prognosis by early detection of relapse. In this study, follow-up data from 72 osteosarcoma patients were received in order to determine the value of clinical examination (CE), lung CT-scan (CTL), chest X-ray (CXR), local X-ray (LXR), and bone scintigraphy (BS) in the detection of tumor recurrence. PROCEDURE: Twenty-eight of 72 osteosarcoma patients presented with a total of 61 relapse sites. A continuous remission after relapse treatment could be achieved in 2/16 patients with first lung metastases, in 2/6 patients with local relapse, and in 3/19 patients with more than one lung metastasis. More than 90% of all relapses occurred within 3 years off primary therapy, respectively, within 3 years after detection of relapse. Local relapse and lung metastases were primarily diagnosed by CXR, CTL and CE. BS was the most important investigation to detect distant metastases. No relapse was found by routine X-ray of the primary tumor site. CONCLUSIONS: To improve efficacy of follow-up programs and to reduce radiation load of nonrelapsed patients, the prognosis of patients with lung metastases or local recurrences and the time of high risk for a relapse should be taken into consideration. Since the number of patients who benefit from relapse therapy is still low, it remains to be shown whether an increased frequency of lung CT-scans or MRIs of the primary tumor site will improve early detection of relapse; and if so, whether that will enhance the chance for successful relapse treatment. CXR, CTL and CE should be performed routinely for at least 3 years after completion of therapy or relapse diagnosis. In contrast, BS and LXR appear not to be useful as routine investigations.

An improved method to correct the QT interval of the electrocardiogram for changes in heart rate.

The adequacy of the Bazett formula to correct for heart rate-induced changes in the QT interval of the electrocardiogram has been frequently questioned. In the present study, a simple linear equation was derived, which in anesthetized dogs corrects more adequately for changes in heart rate than the Bazett formula. Regression analysis of experimental data yielded the following equation: QTc = QT - 0.087 (RR - 1000) = QT - 87 (60/HR - 1). The reliability of this equation was investigated in experiments on anesthetized dogs with different cardioactive drugs with a known mechanism of action.

Visual and computer-based analysis of 24 h sleep-waking patterns in the dog.

Sleep and waking EEG patterns recorded over 24 h periods in 7 adult beagle dogs were studied both visually and by computer. Online analysis was carried out using a mini-computer. The following quantitative data were computed and listed (and/or stored on disc) at 30 sec intervals: power spectrum analysis of one cortical derivation by means of Fast Fourier Transformation, the spectrum being divided in 4 bands (0.5-3.5, 3.5-7.5, 7.5-13.5, 13.5-25 c/sec); the power contained in the 3.5-7.5 c/sec band of the hippocampal derivation; a spindle detection algorithm empirically defined and based on the power contained in the 10.5-14.5 c/sec band of subepochs of 0.8 sec and the background power; average EMG and EOG amplitudes. Plots were made of the quantitative data (on the y axis) showing evolution with time (on the x axis). Comparisons with the y ordinates enabled a visual estimate of stage to be made. Automatic classification was done by means of minimal distance, using visually selected epochs and calculated parameters as references. Visual and computer-based analyses allowed the following patterns to be defined: wakefulness, transitional stage, light slow wave sleep, deep slow wave sleep and REM sleep. Sleep-waking cycles of 20-30 min alternated during the 24 h period with rapid transitions from one stage to another.

Electroencephalographic study of the short-acting hypnotics etomidate and methohexital in dogs.

Beagles, implanted with cortical and subcortical electrodes, were given etomidate i.v. (1 mg/kg) over a period of 10 sec. The effects on the EEG were compared with those obtained with 7 mg/kg of methohexital. Both compounds induced hypnosis for a duration of approximately 8 min. The EEGs showed a remarkable similarity. Visual inspection of the records as well as power spectrum analysis revealed a sustained theta-activity with underlying fast activity. The configuration of the waves was rather sharp. The power obtained after etomidate was, however, 2 to 3 times that obtained after methohexital. When the animals awoke from etomidate-induced hypnosis slow waves appeared and were followed by alpha-activity, whereas after methohexital-hypnosis beta-activity predominated. Etomidate slightly increased heart rate, but respiratory depression was not observed. Methohexital caused pronounced tachycardia and apnoea. In 3 out of 6 dogs methohexital caused myoclonus of the hind legs upon awakening from anaesthesia. Etomidate induced myoclonus in one dog during hypnosis.

On-line computation of cardiac output with the thermodilution method, using a digital minicomputer.

In the present study, several techniques for calculating cardiac output were evaluated in order to find an accurate technique suitable for on-line digital computation. The thermodilution method was evaluated by different calculation techniques and by comparing these techniques with electromagnetic flowmeter values. Comparison of thermal dilution curves, manually calculated by the log-normal and the exponential assumption, showed a correlation coefficient of 0.978 between the two methods, the former values being 11% higher. The exponential method correlated very well with the technique, using a correction factor for injection errors (r = 0.999). Since the exponential technique correlated well with the electromagnetic values (r = 0.84) and since it was less complicated than the technique, using correction factors, this technique was chosen for automation. Comparison of exponential calculated thermodilution values with a digital computer and manually, showed a correlation coefficient of 0.991. Therefore, it was concluded that on-line computation of thermodilution curves improves the applicability of the termodilution techniques as a means of measuring cardiac output.