Correction: Non-Canonical NF-κB Activation and Abnormal B Cell Accumulation in Mice Expressing Ubiquitin Protein Ligase-Inactive c-IAP2.

[This corrects the article DOI: 10.1371/journal.pbio.1000518.].

Structural basis for recognition of diubiquitins by NEMO.

NEMO is the regulatory subunit of the IkappaB kinase (IKK) in NF-kappaB activation, and its CC2-LZ region interacts with Lys63 (K63)-linked polyubiquitin to recruit IKK to receptor signaling complexes. In vitro, CC2-LZ also interacts with tandem diubiquitin. Here we report the crystal structure of CC2-LZ with two dimeric coiled coils representing CC2 and LZ, respectively. Surprisingly, mutagenesis and nuclear magnetic resonance experiments reveal that the binding sites for diubiquitins at LZ are composites of both chains and that each ubiquitin in diubiquitins interacts with symmetrical NEMO asymmetrically. For tandem diubiquitin, the first ubiquitin uses the conserved hydrophobic patch and the C-terminal tail, while the second ubiquitin uses an adjacent surface patch. For K63-linked diubiquitin, the proximal ubiquitin uses its conserved hydrophobic patch, while the distal ubiquitin mostly employs the C-terminal arm including the K63 linkage residue. These studies uncover the energetics and geometry for mutual recognition of NEMO and diubiquitins.

Balance between NF-κB p100 and p52 regulates T cell costimulation dependence.

c-IAP1 and c-IAP2 are ubiquitin protein ligases (E3s) that repress noncanonical NF-κB activation. We have created mice that bear a mutation in c-IAP2 that inactivates its E3 activity and interferes, in a dominant-negative fashion, with c-IAP1 E3 activity (c-IAP2(H570A)). The immune response of these animals was explored by infecting them with the Th1-inducing parasite Toxoplasma gondii. Surprisingly, c-IAP2(H570A) mice succumbed because of T cell production of high levels of proinflammatory cytokines. Unlike naive wild-type (WT) cells, which require signals generated by the TCR and costimulatory receptors to become fully activated, naive c-IAP2(H570A) T cells proliferated and produced high levels of IL-2 and IFN-γ to stimulation via TCR alone. c-IAP2(H570A) T cells had constitutive noncanonical NF-κB activation, and IκB kinase inhibition reduced their proliferation to anti-TCR alone to WT levels but had no effect when costimulation via CD28 was provided. Notably, T cells from nfkb2(-/-) mice, which cannot generate the p52 component of noncanonical NF-κB, were also costimulation independent, consistent with the negative role of this unprocessed protein in canonical NF-κB activation. Whereas T cells from nfkb2(+/-) mice behaved like WT, coexpression of a single copy of c-IAP2(H570A) resulted in cleavage of p100, upregulation of p52, and T cell costimulation independence. Thus, p100 represses and p52 promotes costimulation, and the ratio regulates T cell dependence on costimulatory signals.

[Hippocampal stroke]. / Der hippokampale Schlaganfall.

Unilateral cerebral ischemia of the hippocampus is very rare. This paper reviews the literature and presents the case of a 59-year-old woman with an amnestic syndrome due to a left hippocampal stroke. The patient suffered from retrograde amnesia which was most severe over the 2 days prior to presenting and a slight anterograde amnesia. In addition, a verbal memory disorder was confirmed 1 week after admission by neurological tests. As risk factors, arterial hypertension and a relative hyper-beta lipoproteinemia were found. This case shows that unilateral amnestic stroke, e.g. in the hippocampus region, may be the cause of an amnestic syndrome and should be included in the differential diagnostics.

Sensing of Lys 63-linked polyubiquitination by NEMO is a key event in NF-kappaB activation [corrected].

The transcription factor NF-kappaB is sequestered in the cytoplasm in a complex with IkappaB. Almost all NF-kappaB activation pathways converge on IkappaB kinase (IKK), which phosphorylates IkappaB resulting in Lys 48-linked polyubiquitination of IkappaB and its degradation. This allows migration of NF-kappaB to the nucleus where it regulates gene expression. IKK has two catalytic subunits, IKKalpha and IKKbeta, and a regulatory subunit, IKKgamma or NEMO. NEMO is essential for NF-kappaB activation, and NEMO dysfunction in humans is the cause of incontinentia pigmenti and hypohidrotic ectodermal dysplasia and immunodeficiency (HED-ID). The recruitment of IKK to occupied cytokine receptors, and its subsequent activation, are dependent on the attachment of Lys 63-linked polyubiquitin chains to signalling intermediates such as receptor-interacting protein (RIP). Here, we show that NEMO binds to Lys 63- but not Lys 48-linked polyubiquitin, and that single point mutations in NEMO that prevent binding to Lys 63-linked polyubiquitin also abrogates the binding of NEMO to RIP in tumour necrosis factor (TNF)-alpha-stimulated cells, the recruitment of IKK to TNF receptor (TNF-R) 1, and the activation of IKK and NF-kappaB. RIP is also destabilized in the absence of NEMO binding and undergoes proteasomal degradation in TNF-alpha-treated cells. These results provide a mechanism for NEMO's critical role in IKK activation, and a key to understanding the link between cytokine-receptor proximal signalling and IKK and NF-kappaB activation.

Non-canonical NF-κB activation and abnormal B cell accumulation in mice expressing ubiquitin protein ligase-inactive c-IAP2.

Chromosomal translocations between loci encoding MALT1 and c-IAP2 are common in MALT lymphomas. The resulting fusion proteins lack the c-IAP2 RING domain, the region responsible for its ubiquitin protein ligase (E3) activity. Ectopic expression of the fusion protein activates the canonical NF-κB signaling cascade, but how it does so is controversial and how it promotes MALT lymphoma is unknown. Considering recent reports implicating c-IAP1 and c-IAP2 E3 activity in repression of non-canonical NF-κB signaling, we asked if the c-IAP2/MALT fusion protein can initiate non-canonical NF-κB activation. Here we show that in addition to canonical activation, the fusion protein stabilizes NIK and activates non-canonical NF-κB. Canonical but not non-canonical activation depended on MALT1 paracaspase activity, and expression of E3-inactive c-IAP2 activated non-canonical NF-κB. Mice in which endogenous c-IAP2 was replaced with an E3-inactive mutant accumulated abnormal B cells with elevated non-canonical NF-κB and had increased numbers of B cells with a marginal zone phenotype, gut-associated lymphoid hyperplasia, and other features of MALT lymphoma. Thus, the c-IAP2/MALT1 fusion protein activates NF-κB by two distinct mechanisms, and loss of c-IAP2 E3 activity in vivo is sufficient to induce abnormalities common to MALT lymphoma.

Weighted analysis of 2'-fucosyllactose, 3-fucosyllactose, lacto-N-tetraose, 3'-sialyllactose, and 6'-sialyllactose concentrations in human milk.

Over 150 human milk oligosaccharides (HMOs) have been identified and their concentrations in human milk vary depending on Secretor and Lewis blood group status, environmental and geographical factors, lactation stage, gestational period, and maternal health. Quantitation of HMOs in human milk has been the focus of numerous studies, however, comprehensive and weighted statistical analyses of their levels in human milk are lacking. Therefore, weighted means, standard deviations, medians, interquartile ranges, and 90th percentiles for 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), lacto-N-tetraose (LNT), 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL) were calculated using random sampling and the levels of these HMOs in human milk reported in the literature. Probability distributions of the reported levels were also constructed. Although the levels reported in the published studies varied, the weighted means for 2'-FL, 3-FL, LNT, 3'-SL, and 6'-SL were calculated to be 2.58, 0.57, 0.94, 0.28, and 0.39 g/L, respectively, which are consistent with those that have been previously determined in other systematic analyses. Likely due to the use of weighting, the 90th percentiles were greater than the 95% confidence limits that have been previously calculated. Our study therefore provides accurate and important statistical data to help support the level of appropriate HMO supplementation in infant formula.

Urolithiasis through the ages: data on more than 200,000 urinary stone analyses.

PURPOSE: The incidence and prevalence of urolithiasis are increasing but clinicians also have the impression that gender and age distributions of stone formers are changing. Moreover, regional differences in stone occurrence and composition have been observed. We analyzed such trends based on a large series of urinary stone analyses. MATERIALS AND METHODS: A total of 224,085 urinary stone analyses from 22 German centers were evaluated to determine the incidence of stone composition and identify age and gender distributions from 1977 to 2006. A subset of 58,682 stone analyses from 1993 to 2006 was available to identify regional differences in stone composition in Germany. RESULTS: Calcium containing calculi were most common in each gender. The overall male-to-female ratio of 2.4:1 increased from 1977 (1.86:1) to 2006 (2.7:1). The predominance of male calcium stone formers was even higher among elderly patients with a 3.13:1 ratio at ages 60 to 69. Since 1997, we observed a tendency toward an increasing incidence in middle-aged patients at ages 40 to 49 years. While the rate of infection stones constantly decreased, the incidence of uric acid calculi remained stable with an overall rate of 11.7% in males and 7.0% in females with a peak at higher ages. Cystine stones remained rare at 0.4% in males and 0.7% in females. In terms of regional analyses we noted great variation in stone composition in the 2 genders. Uric acid stones were more common in the eastern and southern regions but infection stones were mostly seen in eastern regions. CONCLUSIONS: In what is to our knowledge the largest series of stone analysis reported to date we identified an age and gender relationship of stone formation and composition. Regional variations are common and underline the influence of living habits, diet and standard of medical care on urinary stone formation.

The CD8+ memory T-cell state of readiness is actively maintained and reversible.

The ability of the adaptive immune system to respond rapidly and robustly upon repeated antigen exposure is known as immunologic memory, and it is thought that acquisition of memory T-cell function is an irreversible differentiation event. In this study, we report that many phenotypic and functional characteristics of antigen-specific CD8 memory T cells are lost when they are deprived of contact with dendritic cells. Under these circumstances, memory T cells reverted from G(1) to the G(0) cell-cycle state and responded to stimulation like naive T cells, as assessed by proliferation, dependence upon costimulation, and interferon-gamma production, without losing cell surface markers associated with memory. The memory state was maintained by signaling via members of the tumor necrosis factor receptor superfamily, CD27 and 4-1BB. Foxo1, a transcription factor involved in T-cell quiescence, was reduced in memory cells, and stimulation of naive CD8 cells via CD27 caused Foxo1 to be phosphorylated and emigrate from the nucleus in a phosphatidylinositol-3 kinase-dependent manner. Consistent with these results, maintenance of G(1) in vivo was compromised in antigen-specific memory T cells in vesicular stomatitis virus-infected CD27-deficient mice. Therefore, sustaining the functional phenotype of T memory cells requires active signaling and maintenance.

3-Dimensional JEG-3 choriocarcinoma cell organoids as a model for trophoblast expansion and differentiation.

JEG-3 choriocarcinoma cells have been widely used as a model for placental trophoblast. Herein, 3-dimensional (3D) JEG-3 organoids (JEG-3-ORGs) were established using a protocol that we recently developed for primary cytotrophoblast organoids (CTB-ORGs). 3D JEG-3-ORGs, cultivated in basic culture medium, rapidly divide and spontaneously undergo differentiation. Under stem cell culture conditions (activation of WNT/EGF signalling and inhibition of TGF-ß signalling) smaller organoids with reduced proliferative capacity were generated specifically abolishing expression of extravillous trophoblast (EVT)-specific genes. Similar to CTB-ORGs, removal of the WNT activator CHIR99021 induced re-expression of these genes in JEG-3-ORGs. Hence, JEG-3-ORGs could be used as a model for directed EVT differentiation.

A randomized phase II study comparing the efficacy and safety of the glyco-optimized anti-EGFR antibody tomuzotuximab against cetuximab in patients with recurrent and/or metastatic squamous cell cancer of the head and neck - the RESGEX study.

BACKGROUND: The aim of the RESGEX study was to compare the efficacy and safety of the anti-epidermal growth factor receptor (anti-EGFR) antibody tomuzotuximab against cetuximab both in combination with chemotherapy in patients with recurrent and/or metastatic squamous cell cancer of the head and neck in the first-line treatment. PATIENTS AND METHODS: In this phase II trial 240 patients were equally randomized for six cycles to receive either tomuzotuximab (initial dose 990 mg then 720 mg) weekly and cisplatin 100 mg/m2 and fluorouracil (5-FU; 1000 mg/m2/day, days 1-4) every 3 weeks or cetuximab (400 mg/m2 subsequent 250 mg/m2) weekly with the same chemotherapeutic backbone followed by antibody maintenance treatment. The primary endpoint was progression-free survival. RESULTS: Median progression-free survival was 6.5 months [95% confidence interval (CI) 5.9-7.9 months] in the tomuzotuximab group and 6.2 months (95% CI 5.8-7.3 months) in the cetuximab group (P = 0.86). The median overall survival (OS) estimate was 11.6 months (95% CI 9.5-17.2 months) in the tomuzotuximab group and 13.8 months (95% CI 12.3-16.4 months) in the cetuximab group (P = 0.96). In an exploratory analysis a small subgroup of p16-positive patients had a significantly longer OS compared with p16-negative patients (hazard ratio 1.860, 95% CI 1.09-3.16, P = 0.02). CONCLUSIONS: The glyco-engineered antibody tomuzotuximab failed to demonstrate improved efficacy with a chemotherapeutic backbone in the first-line treatment of recurrent or metastatic head and neck squamous cell carcinoma. It remains a so far unanswered question whether such antibody would partner better with different drugs such as checkpoint inhibitors.

[Disorders of water and sodium balance in intensive care patients]. / Dysnatriämien bei Intensivpatienten.

Intensive care patients often suffer from hypo- or hypernatremia. These dysnatremias reflect an antidiuretic-hormone (ADH)-related water imbalance and are the result of the underlying disease. However, they are often triggered by drug side effects and exacerbated by an intentional or unintentional sodium imbalance. Dysnatremias are also caused by artificial ventilation; however, the mechanisms behind this are beyond the scope of this article. Considerations regarding etiology, water and sodium balance and, in particular, the variable in urine dilution or concentration, take priority over a brisk normalization of sodium concentration. Therefore, the 3 most important factors are: 1) delivery of water and sodium to the collecting duct; 2) generation and maintenance of an osmotic pressure gradient exerted by solutes present in the renal medullary interstitium; 3) the regulated water permeability of collecting duct cells under the control of antidiuretic hormone. With these, most disorders can already be identified from patient history and simple factors such as body weight and serum and urine osmolality.

Posttranscriptional downregulation of c-IAP2 by the ubiquitin protein ligase c-IAP1 in vivo.

Inhibitor of apoptosis proteins (IAPs) c-IAP1 and c-IAP2 were identified as part of the tumor necrosis factor receptor 2 (TNFR2) signaling complex and have been implicated as intermediaries in tumor necrosis factor alpha signaling. Like all RING domain-containing IAPs, c-IAP1 and c-IAP2 have ubiquitin protein ligase (E3) activity. To explore the function of c-IAP1 in a physiologic setting, c-IAP1-deficient mice were generated by homologous gene recombination. These animals are viable and have no obvious sensitization to proapoptotic stimuli. Cells from c-IAP1(-/-) mice do, however, express markedly elevated levels of c-IAP2 protein in the absence of increased c-IAP2 mRNA. In contrast to reports implicating c-IAPs in the activation of NF-kappaB, resting and cytokine-induced NF-kappaB activation was not impaired in c-IAP1-deficient cells. Transient transfection studies with wild-type and E3-defective c-IAP1 revealed that c-IAP2 is a direct target for c-IAP1-mediated ubiquitination and subsequent degradation, which are potentiated by the adaptor function of TRAF2. Thus, the c-IAPs represent a pair of TNFR-associated ubiquitin protein ligases in which one regulates the expression of the other by a posttranscriptional and E3-dependent mechanism.

A phase I study of PankoMab-GEX, a humanised glyco-optimised monoclonal antibody to a novel tumour-specific MUC1 glycopeptide epitope in patients with advanced carcinomas.

BACKGROUND: A phase I open-label dose-escalation study was conducted to define the safety, tolerability, and pharmacokinetics (PK) of PankoMab-GEX, a glyco-optimised humanised IgG1, with high affinity to a novel tumour-specific glycopeptide epitope of MUC1 (TA-MUC1) with excellent preclinical anti-tumour activity. PATIENTS AND METHODS: Seventy-four patients with advanced TA-MUC1-positive carcinomas received PankoMab-GEX intravenously every 3 (Q3W), 2 (Q2W), or 1 (QW) week in doses of 1-2200 mg in a three-plus-three dose-escalation design until disease progression (NCT01222624). RESULTS: No maximum tolerated dose was reached. Adverse events were mainly mild-to-moderate infusion-related reactions (IRRs) by the first infusion in 45% of patients. Only one dose-limiting toxicity, a grade III IRR, was observed. PankoMab-GEX exhibited linear PK over all doses. Mean terminal half-life was 189 ± 66 h (Q3W), without dose dependency. A target trough level ≥50 µg/mL was reached after one infusion with doses ≥1700 mg Q3W in 80% of patients. Clinical benefit in 60 evaluable patients included one complete response in a patient with ovarian cancer treated 483 d and confirmed disease stabilisation in 19 patients lasting a median (range) of 23 (10-109) weeks. All but two of the patients with clinical benefit had received a compounded total dose ≥700 mg over a 3-week period, including 8 of 12 (67%) patients with ovarian cancer. CONCLUSION: PankoMab-GEX is safe, well tolerated, and showed promising anti-tumour activity in advanced disease. A phase IIb study is ongoing evaluating the efficacy of PankoMab-GEX as a maintenance therapy in advanced ovarian cancer.

Preclinical assessment of a new recombinant ADAMTS-13 drug product (BAX930) for the treatment of thrombotic thrombocytopenic purpura.

UNLABELLED: Essentials ADAMTS-13-deficiency is a cause of thrombotic thrombocytopenic purpura (TTP). Preclinical safety of recombinant human ADAMTS-13 (BAX930) was shown in animal models. Preclinical efficacy of BAX930 was shown in a mouse model of TTP. BAX930 showed advantageous efficacy over fresh frozen plasma, the current standard of care. Click to hear Dr Cataland and Prof. Lämmle present a seminar on Thrombotic Thrombocytopenic Purpura (TTP): new Insights in Pathogenesis and Treatment Modalities. SUMMARY: Background Thrombotic thrombocytopenic purpura (TTP) is a rare blood disorder characterized by microthrombosis in small blood vessels of the body, resulting in a low platelet count. Baxalta has developed a new recombinant ADAMTS-13 (rADAMTS-13) product (BAX930) for on-demand and prophylactic treatment of patients with hereditary TTP (hTTP). Objectives To evaluate the pharmacokinetics, efficacy and safety of BAX930 in different species, by use of an extensive preclinical program. Methods The prophylactic and therapeutic efficacies of BAX930 were tested in a previously established TTP mouse model. Pharmacokinetics were evaluated after single intravenous bolus injection in mice and rats, and after repeated dosing in cynomolgus monkeys. Toxicity was assessed in rats and monkeys, safety pharmacology in monkeys, and local tolerance in rabbits. Results BAX930 was shown to be efficacious, as demonstrated by a stabilized platelet count in ADAMTS-13 knockout mice that were thrombocytopenic when treated. Prophylactic efficacy was dose-dependent and comparable with that achieved by treatment with fresh frozen plasma, the mainstay of hTTP treatment. Therapeutic efficacy was treatment interval-dependent. Safety pharmacology evaluation did not show any deleterious effects of BAX930 on cardiovascular and respiratory functions in monkeys. The compound's pharmacokinetics were similar and dose-proportional in mice, rats, and monkeys. BAX930 was well tolerated in rats, monkeys, and rabbits, even at the highest doses tested. Conclusions These results demonstrate that BAX930 has a favorable preclinical profile, and support the clinical development of rADAMTS-13 for the treatment of hTTP.

Resistance factors in colon cancer tissue and the adjacent normal colon tissue: glutathione S-transferases alpha and pi, glutathione and aldehyde dehydrogenase.

Glutathione S-transferases (GST) alpha and pi, glutathione (GSH) and aldehyde dehydrogenase (ADH) were determined in colorectal cancer tissue specimens and in the adjacent normal colon tissue. The median contents in normal and cancer tissue were 8.1 (2.3-30.3) (5-95% quantiles) and 15.1 (5.3-50.3) microg/mg protein for GST pi (P = 0.035), 0.0 (0.0-1.4) and 0.4 (0.0-3.5) microg/mg protein for GST alpha (P = 0.019), 7.3 (1.3-22.7) and 5.6 (2.3-26.0) microg/mg protein for GSH (P = 0.171) and 30.8 (13.0-42.0) and 23.2 (9.0-32.9) microg/mg protein for ADH (P = 0.0017), respectively. Thus, the mean GST alpha and pi both significantly increased in colon cancer compared to the adjacent normal tissue, which underlines their importance as possible resistance factors. A highly significant correlation was obtained between the GSH content in colon cancer and normal tissue (P = 0.0017). Thus, the constitutive GSH expression seems to be maintained during tumor development. A similar correlation was obtained for ADH (P = 0.0075), but the median ADH was lower in cancer tissue compared to the adjacent normal tissue (P = 0.0017). Contrary to GSH and ADH, GST pi did not correlate between normal and colon cancer tissue. Whereas GSH and ADH correlated in normal colon tissue (P = 0.014), no significant correlation for GSH and ADH was observed in colon cancer tissue (P = 0.109). In conclusion, significant correlations between colon cancer and normal tissue were obtained, suggesting that the expression levels of these resistance factors are maintained during carcinogenesis in most patients.

Time domain analysis of oxygen uptake during pseudorandom binary sequence exercise tests.

Pseudorandom binary sequence (PRBS) exercise tests involve repeated switching between two work rates (WR) according to a computer-generated pattern. This paper presents an approach to analysis of O2 uptake (VO2) in the time domain. First, the autocorrelation function (ACF) of the input WR was recognized to be a triangular-shaped pulse that can be taken to be equivalent to a ramp increase followed by a ramp decrease in WR. Then the cross-correlation function of the input (WR) and the output (VO2) was treated as if it were the response to a triangular-shaped pulse. The cross-correlation function was analyzed by fitting a linear summation of the ramp form of a two-component exponential function to this triangular pulse. VO2 responses of eight subjects were obtained from two different PRBS tests, as well as step changes in WR. The first PRBS test consisted of 15 units, each 30 s in duration. Its ACF had a base width of 60 s. The ramp increase-ramp decrease model fit the data throughout the range of response. The second PRBS test had 63 units, each 5 s in duration; thus its ACF base width was 10 s. Again, the ramp model fit adequately. The data from the second PRBS test could be fit by the impulse form of the two-component exponential equation, although the fit in the first 30 s tended to be poorer. The time constants of VO2 dynamics estimated from step and PRBS tests were not significantly different. PRBS tests can be analyzed in the time domain, and the indicators of system dynamics reflect physiological properties similar to those investigated during step changes in WR.

Polysomnography in drug development.

Polysomnography in drug development is used to detect desirable and undesirable effects of drugs on normal and disturbed sleep. Although this method is essential for the approval of new hypnotic drugs, it is quite often neglected in the development of drugs that show unwanted side effects on normal sleep. In this review, the requirements for qualified polysomnography are described, the strong and weak points of the method are discussed, and its importance for the drug development process is pointed out.