Long-term treatment with ivabradine over 12months in patients with chronic heart failure in clinical practice: Effect on symptoms, quality of life and hospitalizations.

BACKGROUND: Ivabradine is indicated to control heart rate in otherwise optimally treated patients with chronic heart failure (CHF) and reduced ejection fraction. However, data on its effectiveness outside clinical trials and longer-term effects are scarce. METHODS: We performed a prospective cohort study involving 249 German resident cardiologists and analyzed the 1-year effectiveness and safety of ivabradine used in CHF outpatients. Data on symptoms, quality of life, and hospitalizations were collected. RESULTS: In total, 767 CHF patients were enrolled to receive ivabradine twice daily, of whom 684 (90%) were still on ivabradine at study end (mean treatment duration 11.2months). The cohort was representative of CHF patients seen in clinical practice in terms of age, risk factor profile, and comorbidities. Concomitant beta-blocker therapy was prescribed in 497 patients (65%). After one year, compared to baseline, heart rate in ivabradine-treated patients was 16bpm lower. This reduction was associated with a significant improvement in NYHA class, and less frequent signs of decompensation (36% to 8%). The proportion of hospitalized patients within 1year decreased from 23% before treatment, to 5% with ivabradine therapy. These improvements in clinical status were accompanied by a reduction in BNP and an increase in LVEF (+5.1% at 1year). Quality of life was significantly improved in all measured dimensions. Adverse drug reactions were noted in 26 patients (3%), and were in line with the known safety profile of ivabradine. CONCLUSIONS: Ivabradine was effective and well-tolerated in CHF patients seen in clinical practice throughout 1year of treatment.

[Is heart rate adequately controlled in chronic systolic heart failure patients in Germany? results from a nationwide survey (INDICATE)]. / Ist die Herzfrequenz bei Herzinsuffizienz-Patienten in Deutschland ausreichend kontrolliert?

BACKGROUND: Elevated resting heart rate is associated with increased morbidity and mortality in patients with chronic systolic heart failure (CHF). Lowering of heart rate improves cardiovascular outcome in these patients. Therefore, heart rate reduction is an important element of therapeutic management and consistently reflected in current European guidelines for heart failure. Methods: The INDICATE study was initiated as a multicenter nationwide cross-sectional survey aiming to analyze the current quality of care in outpatients with CHF (documented left ventricular systolic dysfunction) in Germany. 20 consecutive patients were to be included in the survey from February until June 2012 by 793 cardiologic private practices. Detailed documentation of each patient was performed using a standardized questionnaire. RESULTS: CHF was known for more than 6 months in 88 % of the 15 148 included patients. Mean heart rate in the study population was 73 ± 13 min⁻¹. In 42 % of patients the heart rate was ≥ 75 min⁻¹. 86 % were treated with betablockers. However, higher doses of betablockers were not associated with lower resting heart rate. 27 % of patients remained on heart rates ≥ 75 min⁻¹ although receiving at least 50 % of betablocker target dose. CONCLUSION: INDICATE reveals a considerable proportion of outpatients with CHF showing an elevated heart rate despite beta blockade - irrespective of applied dose. These results emphasize the importance of optimizing the pharmacological management of resting heart rate according to guidelines in these patients.

Human immunodeficiency virus type 1 induces expression of complement factors in human astrocytes.

Since the brain is separated from the blood immune system by a tight barrier, the brain-resident complement system may represent a central player in the immune defense of this compartment against human immunodeficiency virus (HIV). Chronic complement activation, however, may participate in HIV-associated neurodegeneration. Since the level of complement factors in the cerebrospinal fluid is known to be elevated in AIDS-associated neurological disorders, we evaluated the effect of HIV type 1 (HIV-1) on the complement synthesis of brain astrocytes. Incubation of different astrocytic cell lines and primary astrocytes with HIV-1 induced a marked upregulation of the expression of the complement factors C2 and C3. The synthesis of other secreted or membrane-bound complement proteins was not found to be altered. The enhancement of C3 production was measured both on the mRNA level and as secreted protein in the culture supernatants. HIV-1 laboratory strains as well as primary isolates were capable of inducing C3 production with varied effectiveness. The usage of viral coreceptors by HIV-1 was proved to be a prerequisite for the upregulation of C3 synthesis, which was modulated by the simultaneous addition of cytokines. The C3 protein which is secreted after incubation of the cells with HIV was shown to be biologically active as it can participate in the complement cascade.

Inflammation marker 7,8-dihydroneopterin induces apoptosis of neurons and glial cells: a potential contribution to neurodegenerative processes.

Since neopterin is elevated in the cerebrospinal fluid of patients with inflammatory neurological disorders we investigated the source of neopterin in the brain and a possible contribution of biological active pteridines to the development of brain lesions. Astrocytic, neuronal and microglial cell lines were shown to be negative for neopterin production even after stimulation with interferon-gamma (IFN-gamma) indicating that infiltrating monocytes/macrophages might be responsible for neopterin level in CNS. Whereas neopterin did not affect viability of brain cells, its derivative 7,8-dihydroneopterin (7,8-DHN) induced dose-dependently cellular apoptosis in astrocytes and neurons probably via enhancement of nitric oxide synthase (iNOS) expression. This mechanism might represent a possible link between inflammation in the brain and neurodegeneration.

A 60 kD heat-shock protein-like molecule interacts with the HIV transmembrane glycoprotein gp41.

The heat-shock protein hsp60 is typically found in mitochondria, but, in smaller amounts, also in the cell cytoplasm and associated with the cell membrane. Since heat-shock proteins are known to interact with a variety of molecules and since purified HIV-1 particles were described to contain hsp60 molecules, we tested the possibility that a previously described putative receptor for HIV transmembrane protein gp41 is identical to hsp60. The gp41-binding human protein P62 was purified from H9 and Raji cell lysates by a gp41-coupled affinity column. We could show crossreactivity of both polyclonal and monoclonal anti-hsp60 antibodies with the purified P62. In addition we analyzed binding of P18, a soluble gp41 fragment harboring the extracellular domain (Env aa539-684), to recombinant hsp60. Hsp60 bound well to P18-coated ELISA plates whereas HIV-1 surface protein gp120 induced no binding of hsp60. Preincubation of hsp60 with gp41 abolished the binding. The possible role of this molecule as a cofactor in the pathogenesis of HIV disease is discussed.

Antigenic characterization of HIV-1 gp41 binding proteins.

The envelope protein HIV-1 gp41 has been shown to exert various effects on human T-cells, B-cells and monocytes like inhibition of cell proliferation, modulation of MHC expression and cytokine production. In contrast to gpl20, where several receptor molecules have been identified, the receptor for gp41 is still unknown. Using a sepharose column, coupled with recombinant soluble gp41, (rsgp41; Env amino acids 539-684), five gp41-binding proteins of 37, 45, 50, 62 and 100 kDa had been isolated from lysates of the B-cell line Raji. Two mouse antiserums were generated against the proteins P45 and P62 and were tested against the binding specificity of both antiserums. In Western blot analysis the antiserums recognized two protein bands of 45 and 62 kDa in complete Raji cell lysates, as well as the purified proteins P45 and P62, respectively, but did not show any cross-reaction, indicating that the two proteins do not share any immunological epitopes. Besides, the polyclonal antiserums did not recognize the other gp41-binding proteins P37, P50 and P100. Using the P62 antiserum proteins of the same size as in Raji cell lysates were stained in the lysates of the monocytic cell line U937 and the T-cell line H9, demonstrating distribution of P62 in different blood cells. P45 seems not to be identical to HLA-C which had been shown to bind to gp41. These results indicate, that P45 and P62 are two separate gp41-binding proteins without homology to each other or to the other gp41-binding proteins.

Urokinase plasminogen activator receptor (uPAR; CD87) expression on monocytic cells and T cells is modulated by HIV-1 infection.

The transmembranous urokinase-type plasminogen activator receptor (uPAR; CD87) focuses the formation of active plasmin at the cell surface, thus enhancing directional extracellular proteolysis. Since proteolysis is involved in processes like adhesion, chemotaxis and migration which are important for viral spreading, we investigated the expression of uPAR in HIV-infected cells. Expression of CD87 was upmodulated in U937 monocytic cells as well as in the T cell line H9 and in peripheral blood mononuclear cells (PBMC), both on protein and on mRNA level. This upmodulation was not caused by enhanced mRNA stability but by an enhanced transcriptional rate of the CD87 gene as shown by nuclear run-on analysis. To identify the HIV-responsive element in the CD87 promoter we investigated the promoter activity in U937 and H9 cells at different time points after HIV-infection. Although the transcription of the CD87 gene is higher in HIV-infected cells the promoter activity declines after infection, indicating the presence of an additional regulatory element located upstream of the known promoter sequence or in intron sequences.

HIV-1 gp41 by a common immunological epitope induces increased levels of antibodies against human interferon-beta in HIV-1 positive individuals.

Based on our finding that a similar epitope exists between human IFN-beta (aa128-134) and HIV-1 gp41 (aa586-595), we examined 20 sera from healthy and 20 from HIV-1 infected individuals for IFN-beta antibody levels by ELISA. The levels of anti-IFN-beta antibody in sera from HIV-infected individuals were increased by about 160% in comparison with HIV-negative. We affinity-purified anti-gp41 antibodies from sera of HIV-1-infected individuals using rsgp41-sepharose column. One of three antibodies could recognize human IFN-beta in comparison with antibodies from serum of a healthy individual. A mouse antiserum to human IFN-beta recognized rsgp41 (recombinant soluble gp41 Env amino acid 539-684), while the normal mouse serum (pre-immune serum) did not bind to rspg41. These results indicate that a common immunological epitope exists between human IFN-beta and HIV-1 gp41. The sequence-similarity suggests that this common immunological epitope may be located in the region aa128-134 of human IFN-beta and the immunosuppressive domain (aa583-599) of HIV-1 gp41. The increased levels of antibodies against interferon-beta in HIV-1 positive individuals may be explained by a common immunological epitope on human IFN-beta and HIV-1 gp41.

Aldosterone modulates PNA binding cell isoforms within renal collecting duct epithelium.

To investigate the differentiation of the ampullary collecting duct cells into adult principal and intercalated cells, the embryonic cortex of newborn New Zealand rabbit kidney was isolated and brought in culture. With this culture technique the ampullary cells formed a polarized collecting duct epithelium which was kept under permanent exchange of medium and in the presence of aldosterone, arginine vasopressin and/or insulin. After 14 days of perfusion culture the epithelia showed light and dark cells resembling the principal and intercalated cells of the adult collecting duct. The differentiation from embryonic into adult collecting duct cells was controlled by applying the monoclonal antibody CD 7. Independent of the hormonal treatment all of the epithelial cells matured in culture and expressed the CD 7 antigen. This corresponded with the situation found within the adult kidney, where the CD 7 antigen was localized in all principal and intercalated (IC) cells, whereas the embryonic ampullary epithelium in the neonatal kidney remained negative. A differentiation feature of the beta-type intercalated cell was investigated by labeling the cultured epithelia with peanut agglutinin (PNA). In contrast to the CD 7 antigen the development of PNA binding was highly dependent of time and individual hormone administration. While in control epithelia only 8% of PNA positive cells were found, aldosterone induced epithelia revealed 72% PNA labeled cells. The combination of aldosterone and insulin increased the number of PNA-positive cells to 90%. By scanning electron microscopy it could further be shown that several isoforms of cells were reactive with PNA. Thus, in culture the PNA label is not restricted to the typical beta-type IC cells.

Construction of an apparatus for perfusion cell cultures which enables in vitro experiments under organotypic conditions.

The value of cultured cells in cell biological, pharmaceutical or biotechnological research depends on the degree of terminal cell differentiation. In conventional Petri dishes or tissue culture plates it is often difficult to achieve culture conditions which resemble the in situ situation of intact tissue, as regards optimal cell adhesion, exchange of nutrients and metabolic products. These limitations prompted us to develop simple laboratory tools which optimize the environment of cultured cells. A perfusion apparatus with various culture containers and compatible cell holder sets was constructed which allows the simulation of organotypic conditions. (i) The cells can be kept on individual and interchangeable support materials for an optimal cell attachment. (ii) Culture medium can be perfused during the whole culture period. (iii) One type of the new culture container can be perfused with different media at the apical and basal side of the cells, thus mimicking the organotypic environment that applies for epithelial monolayers. Cell culture experiments with renal collecting duct epithelia exhibited an excellent morphological appearance showing typical features of principal and intercalated cells.