Differential regulations of AQP4 and Kir4.1 by triamcinolone acetonide and dexamethasone in the healthy and inflamed retina.

PURPOSE: Glucocorticoids are used to treat macular edema, although the mechanisms underlying this effect remain largely unknown. The authors have evaluated in the normal and endotoxin-induced uveitis (EIU) rats, the effects of dexamethasone (dex) and triamcinolone acetonide (TA) on potassium channel Kir4.1 and aquaporin-4 (AQP4), the two main retinal Müller glial (RMG) channels controlling retinal fluid movement. METHODS: Clinical as well as relatively low doses of dex and TA were injected in the vitreous of normal rats to evaluate their influence on Kir4.1 and AQP4 expression 24 hours later. The dose-dependent effects of the two glucocorticoids were investigated using rat neuroretinal organotypic cultures. EIU was induced by footpad lipopolysaccharide injection, without or with 100 nM intraocular dex or TA. Glucocorticoid receptor and channel expression levels were measured by quantitative PCR, Western blot, and immunohistochemistry. RESULTS: The authors found that dex and TA exert distinct and specific channel regulations at 24 hours after intravitreous injection. Dex selectively upregulated Kir4.1 (not AQP4) in healthy and inflamed retinas, whereas TA induced AQP4 (not Kir4.1) downregulation in normal retina and upregulation in EIU. The lower concentration (100 nM) efficiently regulated the channels. Moreover, in EIU, an inflammatory condition, the glucocorticoid receptor was downregulated in the retina, which was prevented by intravitreous injections of the low concentration of dex or TA. CONCLUSIONS: The results show that dex and TA are far from being equivalent to modulate RMG channels. Furthermore, the authors suggest that low doses of glucocorticoids may have antiedematous effects on the retina with reduced toxicity.

Detection of micrometastases in sentinel lymph nodes from melanoma patients: direct comparison of multimarker molecular and immunopathological methods.

The technique of sentinel lymph node (SLN) dissection is a reliable predictor of metastatic disease in the lymphatic basin draining the primary melanoma. Reverse transcription-polymerase chain reaction (RT-PCR) is emerging as a highly sensitive technique to detect micrometastases in SLNs, but its specificity has been questioned. A prospective SLN study in melanoma patients was undertaken to compare in detail immunopathological versus molecular detection methods. Sentinel lymphadenectomy was performed on 57 patients, with a total of 71 SLNs analysed. SLNs were cut in slices, which were alternatively subjected to parallel multimarker analysis by microscopy (haematoxylin and eosin and immunohistochemistry for HMB-45, S100, tyrosinase and Melan-A/MART-1) and RT-PCR (for tyrosinase and Melan-A/MART-1). Metastases were detected by both methods in 23% of the SLNs (28% of the patients). The combined use of Melan-A/MART-1 and tyrosinase amplification increased the sensitivity of PCR detection of microscopically proven micrometastases. Of the 55 immunopathologically negative SLNs, 25 were found to be positive on RT-PCR. Notably, eight of these SLNs contained naevi, all of which were positive for tyrosinase and/or Melan-A/MART-1, as detected at both mRNA and protein level. The remaining 41% of the SLNs were negative on both immunohistochemistry and RT-PCR. Analysis of a series of adjacent non-SLNs by RT-PCR confirmed the concept of orderly progression of metastasis. Clinical follow-up showed disease recurrence in 12% of the RT-PCR-positive immunopathology-negative SLNs, indicating that even an extensive immunohistochemical analysis may underestimate the presence of micrometastases. However, molecular analyses, albeit more sensitive, need to be further improved in order to attain acceptable specificity before they can be applied diagnostically.

HIV-1 drug resistance transmission networks in southwest Switzerland.

To determine viral subtypes and resistance mutations to antiretroviral treatment (ART) in untreated HIV-1 acutely infected subjects from Southwest Switzerland. Clinical samples were obtained from the HIV primary infection cohort from Lausanne. Briefly, pol gene was amplified by nested PCR and sequenced to generate a 1?kb sequence spanning protease and reverse transcriptase key protein regions. Nucleotide sequences were used to assess viral genotype and ART resistance mutations. Blood specimens and medical information were obtained from 30 patients. Main viral subtypes corresponded to clade B, CRF02_AG, and F1. Resistant mutations to PIs consisted of L10V and accessory mutations 16E and 60E present in all F1 clades. The NNRTI major resistant mutation 103N was detected in all F1 viruses and in other 2 clades. Additionally, we identified F1 sequences from other 6 HIV infected and untreated individuals from Southwest Switzerland, harboring nucleotide motifs and resistance mutations to ART as observed in the F1 strains from the cohort. These data reveal a high transmission rate (16.6%) for NNRTI resistant mutation 103N in a cohort of HIV acute infection. Three of the 5 resistant strains were F1 clades closely related to other F1 isolates from HIV-1 infection untreated patients also coming from Southwest Switzerland. Overall, we provide strong evidence towards an HIV-1 resistant transmission network in Southwest Switzerland. These findings have relevant implications for the local molecular mapping of HIV-1 and future ART surveillance studies in the region.

Structured RNAs in the ENCODE selected regions of the human genome.

Functional RNA structures play an important role both in the context of noncoding RNA transcripts as well as regulatory elements in mRNAs. Here we present a computational study to detect functional RNA structures within the ENCODE regions of the human genome. Since structural RNAs in general lack characteristic signals in primary sequence, comparative approaches evaluating evolutionary conservation of structures are most promising. We have used three recently introduced programs based on either phylogenetic-stochastic context-free grammar (EvoFold) or energy directed folding (RNAz and AlifoldZ), yielding several thousand candidate structures (corresponding to approximately 2.7% of the ENCODE regions). EvoFold has its highest sensitivity in highly conserved and relatively AU-rich regions, while RNAz favors slightly GC-rich regions, resulting in a relatively small overlap between methods. Comparison with the GENCODE annotation points to functional RNAs in all genomic contexts, with a slightly increased density in 3'-UTRs. While we estimate a significant false discovery rate of approximately 50%-70% many of the predictions can be further substantiated by additional criteria: 248 loci are predicted by both RNAz and EvoFold, and an additional 239 RNAz or EvoFold predictions are supported by the (more stringent) AlifoldZ algorithm. Five hundred seventy RNAz structure predictions fall into regions that show signs of selection pressure also on the sequence level (i.e., conserved elements). More than 700 predictions overlap with noncoding transcripts detected by oligonucleotide tiling arrays. One hundred seventy-five selected candidates were tested by RT-PCR in six tissues, and expression could be verified in 43 cases (24.6%).

Agenesis of human pancreas due to decreased half-life of insulin promoter factor 1.

Neonatal diabetes mellitus can be transient or permanent. The severe form of permanent neonatal diabetes mellitus can be associated with pancreas agenesis. Normal pancreas development is controlled by a cascade of transcription factors, where insulin promoter factor 1 (IPF1) plays a crucial role. Here, we describe two novel mutations in the IPF1 gene leading to pancreas agenesis. Direct sequence analysis of exons 1 and 2 of the IPF1 gene revealed two point mutations within the homeobox in exon 2. Genetic analysis of the parents showed that each mutation was inherited from one parent. Mutations localized in helices 1 and 2, respectively, of the homeodomain, decreased the protein half-life significantly, leading to intracellular IPF1 levels of 36% and 27% of wild-type levels. Both mutant forms of IPF1 were normally translocated to the nucleus, and their DNA binding activity on different known target promoters was similar to that of the wild-type protein. However, transcriptional activity of both mutant IPF1 proteins, alone or in combination with HNF3 beta/Foxa2, Pbx1, or the heterodimer E47-beta 2 was reduced, findings accounted for by decreased IPF1 steady state levels and not by impaired protein-protein interactions. We conclude that the IPF1 level is critical for human pancreas formation.

Blocked autophagy sensitizes resistant carcinoma cells to radiation therapy.

Autophagy or "self eating" is frequently activated in tumor cells treated with chemotherapy or irradiation. Whether autophagy represents a survival mechanism or rather contributes to cell death remains controversial. To address this issue, the role of autophagy in radiosensitive and radioresistant human cancer cell lines in response to gamma-irradiation was examined. We found irradiation-induced accumulation of autophagosomes accompanied by strong mRNA induction of the autophagy-related genes beclin 1, atg3, atg4b, atg4c, atg5, and atg12 in each cell line. Transduction of specific target-siRNAs led to down-regulation of these genes for up to 8 days as shown by reverse transcription-PCR and Western blot analysis. Blockade of each autophagy-related gene was associated with strongly diminished accumulation of autophagosomes after irradiation. As shown by clonogenic survival, the majority of inhibited autophagy-related genes, each alone or combined, resulted in sensitization of resistant carcinoma cells to radiation, whereas untreated resistant cells but not sensitive cells survived better when autophagy was inhibited. Similarly, radiosensitization or the opposite was observed in different sensitive carcinoma cells and upon inhibition of different autophagy genes. Mutant p53 had no effect on accumulation of autophagosomes but slightly increased clonogenic survival, as expected, because mutated p53 protects cells by conferring resistance to apoptosis. In our system, short-time inhibition of autophagy along with radiotherapy lead to enhanced cytotoxicity of radiotherapy in resistant cancer cells.

Surveillance for European bat lyssavirus in Swiss bats.

Most countries in Western Europe are currently free of rabies in terrestrial mammals. Nevertheless, rabies remains a residual risk to public health due to the natural circulation of bat-specific viruses, such as European bat lyssaviruses (EBLVs). European bat lyssavirus types 1 and 2 (EBLV-1 and EBLV-2) are widely distributed throughout Europe, but little is known of their true prevalence and epidemiology. We report that only three out of 837 brains taken from bats submitted to the Swiss Rabies Centre between 1976 and 2009 were found by immunofluorescence (FAT) to be positive for EBLVs. All three positive cases were in Myotis daubentoni, from 1992, 1993 and 2002. In addition to this passive surveillance, we undertook a targeted survey in 2009, aimed at detecting lyssaviruses in live bats in Switzerland. A total of 237 bats of the species M. daubentoni, Myotis myotis, Eptesicus serotinus and Nyctalus noctula were captured at different sites in western Switzerland. Oropharyngeal swabs and blood from each individual were analysed by RT-PCR and rapid fluorescent focus inhibition test (RFFIT), respectively. RNA corresponding to EBLV-2 was detected from oropharyngeal swabs of a single M. daubentoni bat, but no infectious virus was found. Molecular phylogenetic analysis revealed that the corresponding sequence was closely related to the other EBLV-2 sequences identified in previous rabies isolates from Swiss bats (particularly to that found at Geneva in 2002). Three M. daubentoni bats were found to be seropositive by RFFIT. In conclusion, even though the prevalence is low in Switzerland, continuous management and surveillance are required to assess the potential risk to public health.

Impaired glucose homeostasis in mice lacking the alpha1b-adrenergic receptor subtype.

To assess the role of the alpha1b-adrenergic receptor (AR) in glucose homeostasis, we investigated glucose metabolism in knockout mice deficient of this receptor subtype (alpha1b-AR-/-). Mutant mice had normal blood glucose and insulin levels, but elevated leptin concentrations in the fed state. During the transition to fasting, glucose and insulin blood concentrations remained markedly elevated for at least 6 h and returned to control levels after 24 h whereas leptin levels remained high at all times. Hyperinsulinemia in the post-absorptive phase was normalized by atropine or methylatropine indicating an elevated parasympathetic activity on the pancreatic beta cells, which was associated with increased levels of hypothalamic NPY mRNA. Euglycemic clamps at both low and high insulin infusion rates revealed whole body insulin resistance with reduced muscle glycogen synthesis and impaired suppression of endogenous glucose production at the low insulin infusion rate. The liver glycogen stores were 2-fold higher in the fed state in the alpha1b-AR-/- compared with control mice, but were mobilized at the same rate during the fed to fast transition or following glucagon injections. Finally, high fat feeding for one month increased glucose intolerance and body weight in the alpha1b-AR-/-, but not in control mice. Altogether, our results indicate that in the absence of the alpha1b-AR the expression of hypotalamic NPY and the parasympathetic nervous activity are both increased resulting in hyperinsulinemia and insulin resistance as well as favoring obesity and glucose intolerance development during high fat feeding.

The sirtuin inhibitor cambinol impairs MAPK signaling, inhibits inflammatory and innate immune responses and protects from septic shock.

Sirtuins (SIRT1-7) are NAD(+)-dependent histone deacetylases (HDACs) that play an important role in the control of metabolism and proliferation and the development of age-associated diseases like oncologic, cardiovascular and neurodegenerative diseases. Cambinol was originally described as a compound inhibiting the activity of SIRT1 and SIRT2, with efficient anti-tumor activity in vivo. Here, we studied the effects of cambinol on microbial sensing by mouse and human immune cells and on host innate immune responses in vivo. Cambinol inhibited the expression of cytokines (TNF, IL-1β, IL-6, IL-12p40, and IFN-γ), NO and CD40 by macrophages, dendritic cells, splenocytes and whole blood stimulated with a broad range of microbial and inflammasome stimuli. Sirtinol, an inhibitor of SIRT1 and SIRT2 structurally related to cambinol, also decreased macrophage response to TLR stimulation. On the contrary, selective inhibitors of SIRT1 (EX-527 and CHIC-35) and SIRT2 (AGK2 and AK-7) used alone or in combination had no inhibitory effect, suggesting that cambinol and sirtinol act by targeting more than just SIRT1 and SIRT2. Cambinol and sirtinol at anti-inflammatory concentrations also did not inhibit SIRT6 activity in in vitro assay. At the molecular level, cambinol impaired stimulus-induced phosphorylation of MAPKs and upstream MEKs. Going well along with its powerful anti-inflammatory activity, cambinol reduced TNF blood levels and bacteremia and improved survival in preclinical models of endotoxic shock and septic shock. Altogether, our data suggest that pharmacological inhibitors of sirtuins structurally related to cambinol may be of clinical interest to treat inflammatory diseases.

Ectodysplasin has a dual role in ectodermal organogenesis: inhibition of Bmp activity and induction of Shh expression.

Ectodermal organogenesis is regulated by inductive and reciprocal signalling cascades that involve multiple signal molecules in several conserved families. Ectodysplasin-A (Eda), a tumour necrosis factor-like signalling molecule, and its receptor Edar are required for the development of a number of ectodermal organs in vertebrates. In mice, lack of Eda leads to failure in primary hair placode formation and missing or abnormally shaped teeth, whereas mice overexpressing Eda are characterized by enlarged hair placodes and supernumerary teeth and mammary glands. Here, we report two signalling outcomes of the Eda pathway: suppression of bone morphogenetic protein (Bmp) activity and upregulation of sonic hedgehog (Shh) signalling. Recombinant Eda counteracted Bmp4 activity in developing teeth and, importantly, inhibition of BMP activity by exogenous noggin partially restored primary hair placode formation in Eda-deficient skin in vitro, indicating that suppression of Bmp activity was compromised in the absence of Eda. The downstream effects of the Eda pathway are likely to be mediated by transcription factor nuclear factor-kappaB (NF-kappaB), but the transcriptional targets of Edar have remained unknown. Using a quantitative approach, we show in cultured embryonic skin that Eda induced the expression of two Bmp inhibitors, Ccn2/Ctgf (CCN family protein 2/connective tissue growth factor) and follistatin. Moreover, our data indicate that Shh is a likely transcriptional target of Edar, but, unlike noggin, recombinant Shh was unable to rescue primary hair placode formation in Eda-deficient skin explants.

Decreasing population selection rates of resistance mutation K65R over time in HIV-1 patients receiving combination therapy including tenofovir.

OBJECTIVES: The use of tenofovir is highly associated with the emergence of mutation K65R, which confers broad resistance to nucleoside/nucleotide analogue reverse transcriptase inhibitors (NRTIs), especially when tenofovir is combined with other NRTIs also selecting for K65R. Although recent HIV-1 treatment guidelines discouraging these combinations resulted in reduced K65R selection with tenofovir, updated information on the impact of currently recommended regimens on the population selection rate of K65R is presently lacking. METHODS: In this study, we evaluated changes over time in the selection rate of resistance mutation K65R in a large population of 2736 HIV-1-infected patients failing combination antiretroviral treatment between 2002 and 2010. RESULTS: The K65R resistance mutation was detected in 144 patients, a prevalence of 5.3%. A large majority of observed K65R cases were explained by the use of tenofovir, reflecting its wide use in clinical practice. However, changing patterns over time in NRTIs accompanying tenofovir resulted in a persistent decreasing probability of K65R selection by tenofovir-based therapy. The currently recommended NRTI combination tenofovir/emtricitabine was associated with a low probability of K65R emergence. For any given dual NRTI combination including tenofovir, higher selection rates of K65R were consistently observed with a non-nucleoside reverse transcriptase inhibitor than with a protease inhibitor as the third agent. DISCUSSION: Our finding of a stable time trend of K65R despite elevated use of tenofovir illustrates increased potency of current HIV-1 therapy including tenofovir.

Improved virological outcome in White patients infected with HIV-1 non-B subtypes compared to subtype B.

BACKGROUND: Antiretroviral compounds have been predominantly studied in human immunodeficiency virus type 1 (HIV-1) subtype B, but only ~10% of infections worldwide are caused by this subtype. The analysis of the impact of different HIV subtypes on treatment outcome is important. METHODS: The effect of HIV-1 subtype B and non-B on the time to virological failure while taking combination antiretroviral therapy (cART) was analyzed. Other studies that have addressed this question were limited by the strong correlation between subtype and ethnicity. Our analysis was restricted to white patients from the Swiss HIV Cohort Study who started cART between 1996 and 2009. Cox regression models were performed; adjusted for age, sex, transmission category, first cART, baseline CD4 cell counts, and HIV RNA levels; and stratified for previous mono/dual nucleoside reverse-transcriptase inhibitor treatment. RESULTS: Included in our study were 4729 patients infected with subtype B and 539 with non-B subtypes. The most prevalent non-B subtypes were CRF02_AG (23.8%), A (23.4%), C (12.8%), and CRF01_AE (12.6%). The incidence of virological failure was higher in patients with subtype B (4.3 failures/100 person-years; 95% confidence interval [CI], 4.0-4.5]) compared with non-B (1.8 failures/100 person-years; 95% CI, 1.4-2.4). Cox regression models confirmed that patients infected with non-B subtypes had a lower risk of virological failure than those infected with subtype B (univariable hazard ratio [HR], 0.39 [95% CI, .30-.52; P < .001]; multivariable HR, 0.68 [95% CI, .51-.91; P = .009]). In particular, subtypes A and CRF02_AG revealed improved outcomes (multivariable HR, 0.54 [95% CI, .29-.98] and 0.39 [95% CI, .19-.79], respectively). CONCLUSIONS: Improved virological outcomes among patients infected with non-B subtypes invalidate concerns that these individuals are at a disadvantage because drugs have been designed primarily for subtype B infections.

Superinfection with drug-resistant HIV is rare and does not contribute substantially to therapy failure in a large European cohort.

BACKGROUND: Superinfection with drug resistant HIV strains could potentially contribute to compromised therapy in patients initially infected with drug-sensitive virus and receiving antiretroviral therapy. To investigate the importance of this potential route to drug resistance, we developed a bioinformatics pipeline to detect superinfection from routinely collected genotyping data, and assessed whether superinfection contributed to increased drug resistance in a large European cohort of viremic, drug treated patients. METHODS: We used sequence data from routine genotypic tests spanning the protease and partial reverse transcriptase regions in the Virolab and EuResist databases that collated data from five European countries. Superinfection was indicated when sequences of a patient failed to cluster together in phylogenetic trees constructed with selected sets of control sequences. A subset of the indicated cases was validated by re-sequencing pol and env regions from the original samples. RESULTS: 4425 patients had at least two sequences in the database, with a total of 13816 distinct sequence entries (of which 86% belonged to subtype B). We identified 107 patients with phylogenetic evidence for superinfection. In 14 of these cases, we analyzed newly amplified sequences from the original samples for validation purposes: only 2 cases were verified as superinfections in the repeated analyses, the other 12 cases turned out to involve sample or sequence misidentification. Resistance to drugs used at the time of strain replacement did not change in these two patients. A third case could not be validated by re-sequencing, but was supported as superinfection by an intermediate sequence with high degenerate base pair count within the time frame of strain switching. Drug resistance increased in this single patient. CONCLUSIONS: Routine genotyping data are informative for the detection of HIV superinfection; however, most cases of non-monophyletic clustering in patient phylogenies arise from sample or sequence mix-up rather than from superinfection, which emphasizes the importance of validation. Non-transient superinfection was rare in our mainly treatment experienced cohort, and we found a single case of possible transmitted drug resistance by this route. We therefore conclude that in our large cohort, superinfection with drug resistant HIV did not compromise the efficiency of antiretroviral treatment.

Fibroblastic reticular cells in lymph nodes regulate the homeostasis of naive T cells.

Interleukin 7 is essential for the survival of naive T lymphocytes. Despite its importance, its cellular source in the periphery remains poorly defined. Here we report a critical function for lymph node access in T cell homeostasis and identify T zone fibroblastic reticular cells in these organs as the main source of interleukin 7. In vitro, T zone fibroblastic reticular cells were able to prevent the death of naive T lymphocytes but not of B lymphocytes by secreting interleukin 7 and the CCR7 ligand CCL19. Using gene-targeted mice, we demonstrate a nonredundant function for CCL19 in T cell homeostasis. Our data suggest that lymph nodes and T zone fibroblastic reticular cells have a key function in naive CD4(+) and CD8(+) T cell homeostasis by providing a limited reservoir of survival factors.

Antiretroviral treatment of adult HIV infection: 2012 recommendations of the International Antiviral Society-USA panel.

CONTEXT: New trial data and drug regimens that have become available in the last 2 years warrant an update to guidelines for antiretroviral therapy (ART) in human immunodeficiency virus (HIV)-infected adults in resource-rich settings. OBJECTIVE: To provide current recommendations for the treatment of adult HIV infection with ART and use of laboratory-monitoring tools. Guidelines include when to start therapy and with what drugs, monitoring for response and toxic effects, special considerations in therapy, and managing antiretroviral failure. DATA SOURCES, STUDY SELECTION, AND DATA EXTRACTION: Data that had been published or presented in abstract form at scientific conferences in the past 2 years were systematically searched and reviewed by an International Antiviral Society-USA panel. The panel reviewed available evidence and formed recommendations by full panel consensus. DATA SYNTHESIS: Treatment is recommended for all adults with HIV infection; the strength of the recommendation and the quality of the evidence increase with decreasing CD4 cell count and the presence of certain concurrent conditions. Recommended initial regimens include 2 nucleoside reverse transcriptase inhibitors (tenofovir/emtricitabine or abacavir/lamivudine) plus a nonnucleoside reverse transcriptase inhibitor (efavirenz), a ritonavir-boosted protease inhibitor (atazanavir or darunavir), or an integrase strand transfer inhibitor (raltegravir). Alternatives in each class are recommended for patients with or at risk of certain concurrent conditions. CD4 cell count and HIV-1 RNA level should be monitored, as should engagement in care, ART adherence, HIV drug resistance, and quality-of-care indicators. Reasons for regimen switching include virologic, immunologic, or clinical failure and drug toxicity or intolerance. Confirmed treatment failure should be addressed promptly and multiple factors considered. CONCLUSION: New recommendations for HIV patient care include offering ART to all patients regardless of CD4 cell count, changes in therapeutic options, and modifications in the timing and choice of ART in the setting of opportunistic illnesses such as cryptococcal disease and tuberculosis.