Functional analysis of a missense mutation in the serine protease inhibitor SPINT2 associated with congenital sodium diarrhea.

Membrane-bound serine proteases play important roles in different biological processes. Their regulation by endogenous inhibitors is poorly understood. A Y163C mutation in the SPINT2 gene encoding the serine protease inhibitor Hepatocyte Growth Factor Inhibitor HAI-2 is associated with a congenital sodium diarrhea. The functional consequences of this mutation on HAI-2 activity and its physiological targets are unknown. We established a cellular assay in Xenopus laevis oocytes to study functional interactions between HAI-2 and candidate membrane-bound serine proteases expressed in the gastro-intestinal tract. We found that the wild-type form of HAI-2 is a potent inhibitor of nine gastro-intestinal serine proteases. The Y163C mutation in the second Kunitz domain of HAI-2 resulted in a complete loss of inhibitory activity on two intestinal proteases, prostasin and tmprss13. The effect of the mutation of the homologous Y68C in the first Kunitz domain of HAI-2 is consistent with a differential contribution of the two Kunitz domains of HAI-2 in the inhibition of serine proteases. By contrast to the Tyr to Cys, the Tyr to Ser substitution did not change the inhibitory potency of HAI-2, indicating that the thiol-group of the cysteine rather than the Tyr deletion is responsible for the HAI-2 loss of function. Our functional assay allowed us to identify membrane-bound serine proteases as cellular target for inhibition by HAI-2 wild type and mutants, and to better define the role of the Tyr in the second Kunitz domain in the inhibitory activity of HAI-2.

Eosinophilic oesophagitis and proton pump inhibitor-responsive oesophageal eosinophilia have similar clinical, endoscopic and histological findings.

BACKGROUND: Some patients with a phenotypic appearance of eosinophilic oesophagitis (EoE) respond histologically to PPI, and are described as having PPI-responsive oesophageal eosinophilia (PPI-REE). It is unclear if PPI-REE is a GERD-related phenomenon, a subtype of EoE, or a completely unique entity. AIM: To compare demographic, clinical and histological features of EoE and PPI-REE. METHODS: Two databases were reviewed from the Walter Reed and Swiss EoE databases. Patients were stratified into two groups, EoE and PPI-REE, based on recent EoE consensus guidelines. Response to PPI was defined as achieving less than 15 eos/hpf and a 50% decrease from baseline following at least a 6-week course of treatment. RESULTS: One hundred and three patients were identified (63 EoE and 40 PPI-REE; mean age 40.2 years, 75% male and 89% Caucasian). The two cohorts had similar dysphagia (97% vs. 100%, P = 0.520), food impaction (43% vs. 35%, P = 0.536), and heartburn (33% vs. 32%, P = 1.000) and a similar duration of symptoms (6.0 years vs. 5.8 years, P = 0.850). Endoscopic features were also similar between EoE and PPI-REE; rings (68% vs. 68%, P = 1.000), furrows (70% vs. 70%, P = 1.000), plaques (19% vs. 10%, P = 0.272), strictures (49% vs. 30%, P = 0.066). EoE and PPI-REE were similar in the number of proximal (39 eos/hpf vs. 38 eos/hpf, P = 0.919) and distal eosinophils (50 vs. 43 eos/hpf, P = 0.285). CONCLUSIONS: EoE and PPI-responsive oesophageal eosinophilia are similar in clinical, histological and endoscopic features and therefore are indistinguishable without a PPI trial. Further studies are needed to determine why a subset of patients with oesophageal eosinophilia respond to PPI.

Down-regulation of interferon signature in systemic lupus erythematosus patients by active immunization with interferon α-kinoid.

OBJECTIVE: We developed interferon-α-kinoid (IFN-K), a drug composed of inactivated IFNα coupled to a carrier protein, keyhole limpet hemocyanin. In human IFNα-transgenic mice, IFN-K induces polyclonal antibodies that neutralize all 13 subtypes of human IFNα. We also previously demonstrated that IFN-K slows disease progression in a mouse model of systemic lupus erythematosus (SLE). This study was undertaken to examine the safety, immunogenicity, and biologic effects of active immunization with IFN-K in patients with SLE. METHODS: We performed a randomized, double-blind, placebo-controlled, phase I/II dose-escalation study comparing 3 or 4 doses of 30 μg, 60 μg, 120 μg, or 240 μg of IFN-K or placebo in 28 women with mild to moderate SLE. RESULTS: IFN-K was well tolerated. Two SLE flares were reported as serious adverse events, one in the placebo group and the other in a patient who concomitantly stopped corticosteroids 2 days after the first IFN-K dose, due to mild fever not related to infection. Transcriptome analysis was used to separate patients at baseline into IFN signature-positive and -negative groups, based on the spontaneous expression of IFN-induced genes. IFN-K induced anti-IFNα antibodies in all immunized patients. Notably, significantly higher anti-IFNα titers were found in signature-positive patients than in signature-negative patients. In IFN signature-positive patients, IFN-K significantly reduced the expression of IFN-induced genes. The decrease in IFN score correlated with the anti-IFNα antibody titer. Serum complement C3 levels were significantly increased in patients with high anti-IFNα antibody titers. CONCLUSION: These results show that IFN-K is well tolerated, immunogenic, and significantly improves disease biomarkers in SLE patients, indicating that further studies of its clinical efficacy are warranted.

The caspase-8 inhibitor FLIP promotes activation of NF-kappaB and Erk signaling pathways.

BACKGROUND: Activation of Fas (CD95) by its ligand (FasL) rapidly induces cell death through recruitment and activation of caspase-8 via the adaptor protein Fas-associated death domain protein (FADD). However, Fas signals do not always result in apoptosis but can also trigger a pathway that leads to proliferation. We investigated the level at which the two conflicting Fas signals diverge and the protein(s) that are implicated in switching the response. RESULTS: Under conditions in which proliferation of CD3-activated human T lymphocytes is increased by recombinant FasL, there was activation of the transcription factors NF-kappaB and AP-1 and recruitment of the caspase-8 inhibitor and FADD-interacting protein FLIP (FLICE-like inhibitory protein). Fas-recruited FLIP interacts with TNF-receptor associated factors 1 and 2, as well as with the kinases RIP and Raf-1, resulting in the activation of the NF-kappaB and extracellular signal regulated kinase (Erk) signaling pathways. In T cells these two signal pathways are critical for interleukin-2 production. Increased expression of FLIP in T cells resulted in increased production of interleukin-2. CONCLUSIONS: We provide evidence that FLIP is not simply an inhibitor of death-receptor-induced apoptosis but that it also mediates the activation of NF-kappaB and Erk by virtue of its capacity to recruit adaptor proteins involved in these signaling pathways.

Neonatal lupus erythematosus with congenital heart block and severe heart failure due to myocarditis and endocardititis of the mitral valve.

We report a case of neonatal lupus erythematosus (NLE) with congenital heart block and severe myocardial failure, which was followed from the 25th week of gestation because of fetal bradycardia. The child was delivered at the 37th week of gestation by elective cesarean section because of echocardiographically documented heart enlargement, pericardial effusion and moderate insufficiency of the mitral and tricuspid valves. In spite of immediate pacing, intubation and supportive treatment, the newborn developed progressive heart failure. Echocardiography showed endocarditis of the mitral valve and diffuse myocarditis. The heart failure resolved under steroid treatment. Our experience supports the early use of steroids in treating myocarditis due to NLE. Intrauterine steroid treatment in the presence of fetal hydrops and congenital heart block is discussed.

Plasminogen activator inhibitor 1 and plasminogen activator inhibitor 2 in various disease states.

The association of increased PA-inhibitor (PAI) activity and of PAI-1 and PAI-2 antigen levels with different pathological conditions was studied in a collective of over 300 patients. PAI-1 and PAI-2 levels were measured by specific radioimmunoassays. A good correlation was observed of PAI activity with PAI-1 antigen (r = 0.718; p less than 0.0001) but not with PAI-2 (r = 0.070; n.s.). Both in the controls and in the patients, PAI activity and PAI-1 antigen showed an extremely large range of values. PAI activity ranged from 0.5 to 68 U/ml and PAI-1 antigen from 6 to 600 ng/ml. Increased PAI activity and PAI-1 antigen was observed in patients with malignant tumors, cardiovascular or thromboembolic disease, in the postoperative phase, with hepatic insufficiency, after trauma and after extracorporeal circulation. The large spectrum of disease states with increased PAI activity and PAI-1 antigen reinforces previous suggestions that PAI-1 is an acute phase reactant. After extracorporeal circulation, PAI activity and PAI-1 concentrations strongly increased within one hour, remained elevated for at least one week and returned to preoperation values within 7 days. PAI-2 values ranged from below detection limit (15 ng/ml), observed in half of the plasmas, to 485 ng/ml in a pregnant woman. High values of PAI-2 were only observed in pregnancy.

Hemodynamic and humoral effects of the new renin inhibitor enalkiren in normal humans.

The effect of the renin inhibitor enalkiren (Abbott-64662) was evaluated in eight normal volunteer subjects on a standardized sodium diet (100 mmol/day) by measurement of various components of the renin-angiotensin system and drug levels in plasma. On day 1, vehicle and doses of 0.001, 0.003, and 0.01 mg/kg i.v. were administered within 2 minutes at 90-minute intervals. On day 2, vehicle and doses of 0.01, 0.03, and 0.1 mg/kg i.v. were given. With the higher doses, blood pressure tended to decrease slightly with no change in heart rate. Plasma renin activity and plasma angiotensin-(1-8)octapeptide (angiotensin II) fell markedly in a dose-dependent manner. Inhibition of plasma renin activity was maximal 5 minutes after administration of the drug and persisted 90 minutes after the doses of 0.03 and 0.1 mg/kg. Not surprisingly, there was a close correlation between plasma renin activity and plasma angiotensin II levels (r = 0.81, n = 28, p less than 0.001). In contrast, active and total renin measured directly by monoclonal antibodies rose in dose-related fashion in response to renin inhibition. Pharmacokinetic parameters were calculated using the plasma drug concentrations obtained up to 6 hours after the 0.1 mg/kg dose. By means of a two-compartment model, plasma mean half-life of the drug was estimated at 1.60 +/- 0.43 hours.

Fructose Rich Diet-Induced High Plasminogen Activator Inhibitor-1 (PAI-1) Production in the Adult Female Rat: Protective Effect of Progesterone.

The effect of progesterone (P4) on fructose rich diet (FRD) intake-induced metabolic, endocrine and parametrial adipose tissue (PMAT) dysfunctions was studied in the adult female rat. Sixty day-old rats were i.m. treated with oil alone (control, CT) or containing P4 (12 mg/kg). Rats ate Purina chow-diet ad libitum throughout the entire experiment and, between 100 and 120 days of age drank ad libitum tap water alone (normal diet; CT-ND and P4-ND) or containing fructose (10% w/v; CT-FRD and P4-FRD). At age 120 days, animals were subjected to a glucose tolerance test or decapitated. Plasma concentrations of various biomarkers and PMAT gene abundance were monitored. P4-ND (vs. CT-ND) rats showed elevated circulating levels of lipids. CT-FRD rats displayed high (vs. CT-ND) plasma concentrations of lipids, leptin, adiponectin and plasminogen activator inhibitor-1 (PAI-1). Lipidemia and adiponectinemia were high (vs. P4-ND) in P4-FRD rats. Although P4 failed to prevent FRD-induced hyperleptinemia, it was fully protective on FRD-enhanced plasma PAI-1 levels. PMAT leptin and adiponectin mRNAs were high in CT-FRD and P4-FRD rats. While FRD enhanced PMAT PAI-1 mRNA abundance in CT rats, this effect was absent in P4 rats. Our study supports that a preceding P4-enriched milieu prevented the enhanced prothrombotic risk induced by FRD-elicited high PAI-1 production.

Angiotensin converting enzyme inhibitor induced angio-oedema: a review of the pathophysiology and risk factors.

Angio-oedema (AE) is a known adverse effect of angiotensin converting enzyme inhibitor (ACE-I) therapy. Over the past several decades, evidence of failure to diagnose this important and potentially fatal reaction is commonly found in the literature. Because this reaction is often seen first in the primary care setting, a review was undertaken to analyse and document the keys to both diagnostic criteria as well as to investigate potential risk factors for ACE-I AE occurrence. A general review of published literature was conducted through Medline, EMBASE, and the Cochrane Database, targeting ACE-I-related AE pathomechanism, diagnosis, epidemiology, risk factors, and clinical decision making and treatment. The incidence and severity of AE appears to be on the rise and there is evidence of considerable delay in diagnosis contributing to significant morbidity and mortality for patients. The mechanism of AE due to ACE-I drugs is not fully understood, but some genomic and metabolomic information has been correlated. Additional epidemiologic data and clinical treatment outcome predictors have been evaluated, creating a basis for future work on the development of clinical prediction tools to aid in risk identification and diagnostic differentiation. Accurate recognition of AE by the primary care provider is essential to limit the rising morbidity associated with ACE-I treatment-related AE. Research findings on the phenotypic indicators relevant to this group of patients as well as basic research into the pathomechanism of AE are available, and should be used in the construction of better risk analysis and clinical diagnostic tools for ACE-I AE.

A peptide inhibitor of c-Jun N-terminal kinase protects against both aminoglycoside and acoustic trauma-induced auditory hair cell death and hearing loss.

Hearing loss can be caused by a variety of insults, including acoustic trauma and exposure to ototoxins, that principally effect the viability of sensory hair cells via the MAP kinase (MAPK) cell death signaling pathway that incorporates c-Jun N-terminal kinase (JNK). We evaluated the otoprotective efficacy of D-JNKI-1, a cell permeable peptide that blocks the MAPK-JNK signal pathway. The experimental studies included organ cultures of neonatal mouse cochlea exposed to an ototoxic drug and cochleae of adult guinea pigs that were exposed to either an ototoxic drug or acoustic trauma. Results obtained from the organ of Corti explants demonstrated that the MAPK-JNK signal pathway is associated with injury and that blocking of this signal pathway prevented apoptosis in areas of aminoglycoside damage. Treatment of the neomycin-exposed organ of Corti explants with D-JNKI-1 completely prevented hair cell death initiated by this ototoxin. Results from in vivo studies showed that direct application of D-JNKI-1 into the scala tympani of the guinea pig cochlea prevented nearly all hair cell death and permanent hearing loss induced by neomycin ototoxicity. Local delivery of D-JNKI-1 also prevented acoustic trauma-induced permanent hearing loss in a dose-dependent manner. These results indicate that the MAPK-JNK signal pathway is involved in both ototoxicity and acoustic trauma-induced hair cell loss and permanent hearing loss. Blocking this signal pathway with D-JNKI-1 is of potential therapeutic value for long-term protection of both the morphological integrity and physiological function of the organ of Corti during times of oxidative stress.

Can resistance to trastuzumab be reversed by endocrine therapy? Combination of trastuzumab and letrozole after resistance to sequential trastuzumab and aromatase inhibitor monotherapies in patients with ER-positive, HER-2 positive, advanced breast cancer: a proof-of-concept trial (SAKK 23/03)

Introduction: Trastuzumab (T) is a cornerstone in the treatment of patients with HER2-overexpressing advanced breast cancer and development of resistance to T is a major therapeutic problem. HER-2 is part of a highly interactive signaling network that may impair efficacy of endocrine therapy. A sequential treatment design was chosen in this trial to ensure complete resistance to single agent therapy before receiving both a non-steroidal aromatase inhibitor (AI) and T. Any kind of clinical activity with combined treatment of AI and T after progression of single agent treatments could indicate restoration of sensitivity as a consequence of cross-talking and networking between both pathways. Methods: Key eligibility criteria included postmenopausal patients (pts.) with advanced, measurable, HER-2 positive (assessed by FISH, ratio (≥2)), HR positive disease and progression on prior treatment with a non-steroidal AI, e.g. letrozole or anastrozole, either in an adjuvant or advanced setting. Pts. received standard dose T monotherapy either weekly or three-weekly in step 1 and upon disease progression, continued T in combination with letrozole in step 2. The primary endpoint was clinical benefit response (CBR: CR, PR or SD for at least 24 weeks (+/- 1 week) according to RECIST) in step 2. Results: Thirteen pts. were enrolled in five centers in Switzerland. In step 1, six pts. (46%) achieved CBR. Median time to progression (TTP) was 161 days (Range: 50 - 627). Based on data collected until the end of May 2010, CBR was observed in seven out of the eleven evaluable pts. (64%) in step 2, including one pt. with partial response. Four of the seven pts. within step 2 that achieved CBR also had CBR in step 1. Seven out of eleven pts. have documented tumor progression during step 2 treatment. Median TTP for all eleven pts. was 184 days (range 61 - 471). Mean time on study treatment (TTP in step 1 plus TTP in step 2) for pts. reaching step 2 was 380 days (range 174 - 864). Adverse events were generally mild. Conclusion: Results of this proof-of-principle trial suggest that complete resistance to both AI and T can be overcome in a proportion of pts. by combined treatment of AI and T, as all pts. served as their own control. Our results appear promising for a new treatment strategy which offers a chemotherapy-free and well-tolerated option for at least a subset of the pts. with HR positive, HER-2 positive breast cancer. Further trials will need to corroborate this finding.

Experimental and clinical radiofrequency ablation: proposal for standardized description of coagulation size and geometry.

BACKGROUND: Radiofrequency (RF) ablation is used to obtain local control of unresectable tumors in liver, kidney, prostate, and other organs. Accurate data on expected size and geometry of coagulation zones are essential for physicians to prevent collateral damage and local tumor recurrence. The aim of this study was to develop a standardized terminology to describe the size and geometry of these zones for experimental and clinical RF. METHODS: In a first step, the essential geometric parameters to accurately describe the coagulation zones and the spatial relationship between the coagulation zones and the electrodes were defined. In a second step, standard terms were assigned to each parameter. RESULTS: The proposed terms for single-electrode RF ablation include axial diameter, front margin, coagulation center, maximal and minimal radius, maximal and minimal transverse diameter, ellipticity index, and regularity index. In addition a subjective description of the general shape and regularity is recommended. CONCLUSIONS: Adoption of the proposed standardized description method may help to fill in the many gaps in our current knowledge of the size and geometry of RF coagulation zones.

Relation between sequence and structure of HIV-1 protease inhibitor complexes: a model system for the analysis of protein flexibility.

The flexibility of different regions of HIV-1 protease was examined by using a database consisting of 73 X-ray structures that differ in terms of sequence, ligands or both. The root-mean-square differences of the backbone for the set of structures were shown to have the same variation with residue number as those obtained from molecular dynamics simulations, normal mode analyses and X-ray B-factors. This supports the idea that observed structural changes provide a measure of the inherent flexibility of the protein, although specific interactions between the protease and the ligand play a secondary role. The results suggest that the potential energy surface of the HIV-1 protease is characterized by many local minima with small energetic differences, some of which are sampled by the different X-ray structures of the HIV-1 protease complexes. Interdomain correlated motions were calculated from the structural fluctuations and the results were also in agreement with molecular dynamics simulations and normal mode analyses. Implications of the results for the drug-resistance engendered by mutations are discussed briefly.