[Severe lymphopenia in a patient with Crohn's disease]. / Schwere Lymphozytopenie bei Patientin mit Morbus Crohn.

We report on a 25-year-old female patient with Crohn's disease and profound lymphocytopenia while receiving corticosteroids and azathioprine. Discontinuation of azathioprine only resulted in a mild increase in CD4+ T cell numbers; however, therapy with the TNFα inhibitor adalimumab was initiated for a clinical flare and resulted in long-lasting clinical remission and rapid normalization of the lymphocytopenia including the respective lymphocyte subsets. Lymphocytopenia is frequently observed as a side effect of immunosuppressive therapy. This case illustrates that lymphocytopenia may also occur in relation to Crohn's disease activity as an extraintestinal manifestation and may then be efficiently treated by escalation of immunosuppressive therapy.

A new functional assay for the diagnosis of X-linked inhibitor of apoptosis (XIAP) deficiency.

X-linked inhibitor of apoptosis (XIAP) deficiency, caused by mutations in BIRC4, is an immunodeficiency associated with immune dysregulation and a highly variable clinical presentation. Current diagnostic screening tests such as flow cytometry for XIAP expression or lymphocyte apoptosis assays have significant limitations. Based on recent evidence that XIAP is essential for nucleotide-binding and oligomerization domains (NOD)1/2 signalling, we evaluated the use of a simple flow cytometric assay assessing tumour necrosis factor (TNF) production of monocytes in response to NOD2 stimulation by muramyl dipeptides (L18-MDP) for the functional diagnosis of XIAP deficiency. We investigated 12 patients with XIAP deficiency, six female carriers and relevant disease controls. Irrespective of the diverse clinical phenotype, the extent of residual protein expression or the nature of the mutation, the TNF response was severely reduced in all patients. On average, L18-MDP induced TNF production in 25% of monocytes from healthy donors or female carriers, while fewer than 6% of monocytes responded in affected patients. Notably, the assay clearly discriminated affected patients from disease controls with other immunodeficiencies accompanied by lymphoproliferation, hypogammaglobulinaemia or inflammatory bowel disease. Functional testing of the NOD2 signalling pathway is an easy, fast and reliable assay in the diagnostic evaluation of patients with suspected XIAP deficiency.

Transfusion-transmitted hepatitis E in Germany, 2013.

The reported IgG seroprevalence against hepatitis E virus (HEV) in German blood donations is 6.8%, and HEV RNA detected in 0.08%, but documented evidence for HEV transmission is lacking. We identified two donations from a single donor containing 120 IU HEV RNA/mL plasma and 490 IU/mL. An infectious dose of 7,056 IU HEV RNA was transmitted via apheresis platelets to an immunosuppressed patient who developed chronic HEV. Further, transmission was probable in an immunocompetent child.

High-dimensional profiling reveals Tc17 cell enrichment in active Crohn's disease and identifies a potentially targetable signature.

The immune-pathology in Crohn's disease is linked to dysregulated CD4+ T cell responses biased towards pathogenic TH17 cells. However, the role of CD8+ T cells able to produce IL-17 (Tc17 cells) remains unclear. Here we characterize the peripheral blood and intestinal tissue of Crohn's disease patients (n = 61) with flow and mass cytometry and reveal a strong increase of Tc17 cells in active disease, mainly due to induction of conventional T cells. Mass cytometry shows that Tc17 cells express a distinct immune signature (CD6high, CD39, CD69, PD-1, CD27low) which was validated in an independent patient cohort. This signature stratifies patients into groups with distinct flare-free survival associated with differential CD6 expression. Targeting of CD6 in vitro reduces IL-17, IFN-γ and TNF production. These results identify a distinct Tc17 cell population in Crohn's disease with proinflammatory features linked to disease activity. The Tc17 signature informs clinical outcomes and may guide personalized treatment decisions.

The transcription factor c-JUN/AP-1 promotes HBV-related liver tumorigenesis in mice.

Hepatocellular carcinoma (HCC) develops as a consequence of chronic inflammatory liver diseases such as chronic hepatitis B virus (HBV) infection. The transcription factor c-Jun/activator protein 1 (AP-1) is strongly expressed in response to inflammatory stimuli, promotes hepatocyte survival during acute hepatitis and acts as an oncogene during chemically induced liver carcinogenesis in mice. Here, we therefore aimed to characterize the functions of c-Jun during HBV-related liver tumorigenesis. To this end, transgenic mice expressing all HBV envelope proteins (HBV(+)), an established model of HBV-related HCC, were crossed with knockout mice lacking c-Jun specifically in hepatocytes and tumorigenesis was analyzed. Hepatic expression of c-Jun was strongly induced at several time points during tumorigenesis in HBV(+) mice, whereas expression of other AP-1 components remained unchanged. Importantly, formation of premalignant foci and tumors was strongly reduced in HBV(+) mice lacking c-Jun. This phenotype correlated with impaired hepatocyte proliferation and increased expression of the cell cycle inhibitor p21, whereas hepatocyte survival was not affected. Progression and prognosis of HBV-related HCC correlates with the expression of the cytokine osteopontin (Opn), an established AP-1 target gene. Opn expression was strongly reduced in HBV(+) livers and primary mouse hepatocytes lacking c-Jun, demonstrating that c-Jun regulates hepatic Opn expression in a cell-autonomous manner. These findings indicate that c-Jun has important functions during HBV-associated tumorigenesis by promoting hepatocyte proliferation as well as progression of dysplasia. Therefore, targeting c-Jun may be a useful strategy to prevent hepatitis-associated tumorigenesis.

Remission of refractory Crohn's disease by high-dose cyclophosphamide and autologous peripheral blood stem cell transplantation.

BACKGROUND: Despite advances in immunosuppressive therapy, up to 10% of patients with severe Crohn's disease (CD) remain refractory to conventional treatment. Limited evidence from pilot trials suggests that high-dose immunosuppression and autologous peripheral blood stem cell transplantation (autoPBSCT) may induce remission in these patients, but there is substantial controversy regarding the safety and efficacy of this approach. AIM: To address this issue, a monocentre phase I/II trial of autoPBSCT was performed in patients with refractory CD in our hospital. METHODS: Here, we report on the outcome of 12 patients with refractory CD treated with autoPBSCT. Briefly, CD34(+) -selected PBSCs were harvested after mobilisation therapy with cyclophosphamide and granulocyte-colony stimulating factor. Later, immunoablative conditioning therapy with high-dose cyclophosphamide followed by autoPBSCT was applied and clinical and endoscopic responses were analysed after a mean follow-up of 3.1 years (range 0.5-10.3 years). RESULTS: PBSC harvest following mobilisation chemotherapy was successful in 11/12 patients and resulted in a clinical and endoscopic improvement in 7/12 patients. Subsequent conditioning and autoPBSCT were performed in nine patients and were relatively well tolerated. Among those, five patients achieved a clinical and endoscopic remission within 6 months after autoPBSCT. However, relapses occurred in 7/9 patients during follow-up, but disease activity could be controlled by low-dose corticosteroids and conventional immunosuppressive therapy. CONCLUSION: Immunoablation by cyclophosphamide and autologous peripheral blood stem cell transplantation is safe and effective to induce remission of refractory Crohn's disease, and should be further evaluated in randomised controlled trials.

The role of the transcription factor AP-1 in colitis-associated and beta-catenin-dependent intestinal tumorigenesis in mice.

Chronic inflammation is an important cancer risk factor but the molecular pathways linking inflammation and cancer are incompletely understood. The transcription factor c-Jun/AP-1 (activator protein 1) is involved in inflammatory responses and tumorigenesis and has been proposed as an essential mediator of oncogenic beta-catenin signaling in the intestine. Here, we examined the functions of c-Jun in two distinct mouse models of conditional and intestine-specific activation of beta-catenin. c-Jun is strongly expressed in the small intestine of mutant mice. However, beta-catenin-dependent cell proliferation is surprisingly not affected in mice lacking c-jun in intestinal epithelium, suggesting that c-Jun is not an essential immediate target of beta-catenin signaling in the small intestine. To examine the functions of Jun and Fos proteins during inflammation and cancer in the colon, colitis-associated tumors were induced chemically in the respective knockout mice. Tumors were characterized by activated beta-catenin and strongly expressed c-Jun and JunB. However, tumorigenesis was not affected by inactivation of c-Jun in either intestinal epithelium or myeloid cells. Moreover, tumorigenesis was not altered in mice lacking junB, junD, c-fos, fra-1 or fra-2, suggesting that inhibition of c-Jun or other single AP-1 proteins is not a determining factor in colitis-associated cancer in mice.

pH regulation in isolated in vitro perfused rat colonic crypts.

We investigated disorders and regulation of cytosolic pH (pHi) in isolated perfused crypts from rat distal colon using the pH-sensitive dye BCECF. This preparation allows distinct examination of either luminal or basolateral transport. The effects of luminal weak organic acids and bases on pHi were examined. The physiological concentrations of both luminal CO2/HCO3- and acetic acid/acetate acidified pHi significantly, but less than when applied from the basolateral side. Corresponding changes (luminal versus basolateral) in pHi were -0.17+/-0.04 versus -0.39+/-0.04, (n=8) and -0.15+/-0.02 versus -0.41+/-0.04, (n=8), respectively. Basolateral versus luminal application of NH3/NH4+ led to a more marked change in pHi, namely 0.35+/-0.03 versus 0.008+/-0.007 pH units, (n=19). The luminal perfusion of NH3/NH4+ was controlled by applying fura-2 acid to the luminal side and at the same time recording fura-2-specific fluorescence. Hence, the influence of luminal acid/base on colonic pHi homeostasis was limited. To examine pHi regulation, we investigated the recovery from an intracellular acid load using the NH3/NH4+ pulse method. Recovery was completely dependent on basolateral Na+, indicating that luminal acid/base transport does not play a major role in pHi homeostasis. The basolateral transporters involved in pHi recovery are probably the EIPA- and HOE694-inhibitable (IC50=0.2 and 2 micromol/l, respectively) Na+/H+ exchanger NHE1 and the DIDS-inhibitable Na+-dependent HCO3- importer.

Rescue of the mineralocorticoid receptor knock-out mouse.

The mineralocorticoid receptor knock-out mouse (MR-/-), resembling inborn pseudohypoaldosteronism, dies 8-12 days after birth in circulatory failure with all the signs of terminal volume contraction. The present study aimed to examine the functional defects in the kidney and colon in detail and to attempt to rescue these mice. In neonatal (nn) MR-/- the amiloride-sensitive short-circuit current in the colon was reduced to approximately one-third compared to controls (MR+/+ and MR+/-). In isolated in vitro perfused collecting ducts the amiloride-induced hyperpolarization of the basolateral membrane (Vbl) of nn MR-/- was similar to that of controls, but urinary Na+ excretion was markedly increased to 4.3 micromol/day.g (BW). Based on this measured urinary Na+ loss we tried to rescue nn MR-/- mice by injecting NaCl twice daily (3.85 micromol/g BW), corresponding to 22 microliter of isotonic saline/g BW subcutaneously. This regimen was continued until the animals had reached a body mass of 8.5 g. Thereafter, in addition to normal chow and tap water, NaCl drinking water (333 mmol/l) and pellets soaked in 333 mmol/l NaCl were offered. Unlike the untreated nn MR-/- most of these mice survived. The adult animals were examined between days 27 and 41, some were used for breeding. When compared to age-matched controls the growth of MR-/- was delayed until day 20. Then their growth curve increased in slope and reached that of controls. MR-/- retained their Na+-losing defect. Amiloride's effect on urinary Na+ excretion was not significant in MR-/- mice and the effect on Vbl in isolated cortical collecting ducts was attenuated. The renin-producing cells were hypertrophic and hyperplastic. Plasma renin and aldosterone concentrations were significantly elevated in MR-/- mice. These data indicate that MR-/- can be rescued by timely and matched NaCl substitutions. This enables the animals to develop through a critical phase of life, after which they adapt their oral salt and water intake to match the elevated excretion rate; however, the renal salt-losing defect persists.

Generation of stable mRNA fragments and translation of N-truncated proteins induced by antisense oligodeoxynucleotides.

Binding of phosphorothioate-modified antisense oligodeoxynucleotides (AS ODNs) to target mRNAs is commonly thought to mediate RNA degradation or block of translation. Here we demonstrate cleavage of target mRNAs within the AS ODN binding region with subsequent degradation of the 5' but not the 3' cleavage product. Some, if not all, 3' mRNA fragments lacked a 5' cap structure, whereas their poly(A) tail length remained unchanged. Furthermore, they were efficiently translated into N-terminally truncated proteins as demonstrated in three settings: production of shortened hepadnaviral surface proteins, alteration of the subcellular localization of a fluorescent protein, and shift of the transcription factor C/EBPalpha isoform expression levels. Thus, AS treatment may result in the synthesis of N-truncated proteins with biologically relevant effects.