Novel Synthetic,Salt-Resistant Analogs of Human Beta-Defensins 1 and 3 Endowed with Enhanced Antimicrobial Activity

Human beta-defensins (hBDs) are antimicrobial peptides of human innate immunity. The antibacterial activities of hBDs 1, 2, and 4 but not the activity of hBD3 are impaired by high salt levels. We have designed and synthesized seven novel hBD analogs, constituted by different domains of hBD1 (which is constitutively expressed in humans) and of hBD3 (which is induced by microorganisms and inflammatory factors in humans), that would maintain and potentially increase the wild-type antimicrobial activities and be salt resistant. We have compared the antibacterial, antiviral, and chemotactic activities of the analogs with those of hBD1 and hBD3. We show that the hBD1 internal region and the hBD3 C-terminal region are critical for antibacterial activity also at high salt concentrations, whereas deletion of the N-terminal region of hBD3 results in an increase in antibacterial activity. All analogs inhibited herpes simplex virus; antiviral activity was enhanced by the hBD1 internal region and the hBD3 C-terminal region. Wild-type and analog peptides were chemotactic for granulocytes and monocytes, irrespective of the salt concentrations. These new peptides may have therapeutic potential.Beta-defensins (BDs) are highly conserved small peptides produced by plants, invertebrates, and vertebrates that developed as part of the primordial immune protective mechanism (19). Four of these peptides, called human BD1 (hBD1; DEFB1), hBD2 (DEFB4), hBD3 (DEFB103A), and hBD4 (DEFB104), are mainly expressed by respiratory, gastrointestinal, and urogenital epithelial cells either constitutively (hBD1) or after induction by microorganisms or inflammatory factors (hBD2 to hBD4) (19). All four hBDs are cationic and 36 to 45 amino acids long and show similar folding and an invariable six-cysteine motif that gives rise to three disulfide bonds (2, 11, 12, 25, 26).Human beta-defensins 1 to 4 exert different bactericidal and antiviral activities against various pathogens (8, 15, 27). The antibacterial effects of hBD1 (9), hBD2 (33), and hBD4 (5) are attenuated by high NaCl concentrations, such as those in the airway surface fluid of patients with cystic fibrosis (CF) (21, 29). Human beta-defensin 3 can withstand NaCl concentrations as high as 150 mM, thanks to its peculiar structural characteristics and charge (10). In the field of viral diseases, hBD2 and -3 inhibit human immunodeficiency virus (HIV) type 1 (HIV-1) replication and virion infectivity (20, 31) and modulate HIV-1 coreceptor expression (20). Human herpes simplex virus (HSV) type 1 (HSV-1), HSV-2, and other viruses preincubated with alpha human neutrophil peptide 1 (hNP1) to hNP3 (6, 28) or theta (37) defensins lose their ability to infect target cells (28). As yet, there are no data on the effect of hBDs on HSV-1 and -2. In addition to direct antimicrobial activity, hBDs also exert chemotactic activity: hBD1, -2, and -3 are chemotactic for monocytes and dendritic and T cells. Human beta-defensin 3 is the only beta-defensin chemotactic for macrophages (4, 18, 19), whereas the chemotactic effect of hBDs on granulocytes has yet to be elucidated (4, 18).The two natural defensins hBD1 and hBD3 were chosen for use in the experiments described in this paper for the following reasons: hBD1 is constitutively expressed but its antibacterial activity is greatly impaired by NaCl, while hBD3 is insensitive to salt. Thus, we designed and synthesized hBD analogs that, in principle, would maintain the antibacterial and antiviral activities of hBD1 and possess a resistance capability in the presence of high NaCl concentrations, like hBD3 does. We then compared the antibacterial, chemotactic, and antiviral activities of the novel synthetic analogs with those of wild-type hBD1 and hBD3. Our data show that some of the synthetic analogs have higher antimicrobial activity than the wild type, also at high NaCl concentrations.     

Post-splenectomy and hyposplenic states

The spleen is crucial in regulating immune homoeostasis through its ability to link innate and adaptive immunity and in protecting against infections. The impairment of splenic function is defined as hyposplenism, an acquired disorder caused by several haematological and immunological diseases. The term asplenia refers to the absence of the spleen, a condition that is rarely congenital and mostly post-surgical. Although hyposplenism and asplenia might predispose individuals to thromboembolic events, in this Review we focus on infectious complications, which are the most widely recognised consequences of these states. Because of the high mortality, the fulminant course, and the refractoriness to common treatment of overwhelming infections caused by encapsulated bacteria, prevention through vaccination and antibiotic prophylaxis is the basis of the management of patients who have had splenectomy or have hyposplenism. In this Review, we critically assess clinical and diagnostic aspects of splenic dysfunction and highlight new perspectives in the prevention of overwhelming post-splenectomy infections.     

Integrated backscatter analysis of carotid intima–media complex in patients with systemic lupus erythematosus

A number of studies based on conventional ultrasound scanning (CUS) gave contrasting results about the occurrence of early atherosclerosis in patients with systemic lupus erythematosus (SLE), while no study on early arterial sclerosis in the same patients are available. Recently, information on early arterial sclerosis can be provided by the integrated backscatter (IBS) analysis which reflects the collagen and calcium content within the vascular wall. In order to evaluate if atherosis and/or sclerosis of carotid arteries are early features of SLE, we performed carotid CUS and IBS analysis in 16 SLE patients (15 females; aged 37 ± 10 years), free from clinically evident cardiovascular diseases and cardiovascular risk factors, with the only exception of five patients who had arterial hypertension. The same investigations were performed in 16 sex- and age-matched healthy control subjects. No statistically significant difference was observed either in carotid corrected IBS values or in carotid intima–media thickness (IMT) values between SLE patients and control subjects (−17.9 ± 2.5 dB vs −19.0 ± 1.7 dB, p = 0.14; 0.66 ± 0.08 mm vs 0.62 ± 0.13 mm, p = 0.35, respectively). The little sub-group of hypertensive SLE patients exhibited a significantly higher carotid corrected IBS mean value compared to control subjects (−16.4 ± 3.1 dB vs −19.0 ± 1.7 dB, p = 0.026), while it did not significantly differ in carotid IMT value from control group (0.67 ± 0.09 mm vs 0.62 ± 0.13 mm, respectively; p = 0.86). These findings show that neither atherosis nor sclerosis of carotid arteries are early features of SLE patients free from cardiovascular risk factors. Further studies are needed to clearly demonstrate that early carotid sclerosis affects hypertensive SLE patients.     

Comparison between neomycin and lactulose in 173 patients with hepatic encephalopathy

A randomized study was performed in order to compare the course of hepatic encephalopathy in patients treated with neomycin plus magnesium sulfate or with lactulose. Admission criteria were: morphological diagnosis of cirrhosis and absence of comorbidity, of contraindications to drugs, or of previous treatments which could influence the outcome. The treatment groups were similar in terms of clinical characteristics, fatalities, recovery rate from grade 1 encephalopathy, and disappearance rate of neuropsychiatric signs. Transitions from severe to grade 1 or 0 encephalopathy showed a 0.17 (NS) difference in favor of neomycin. Early therapy and evidence of precipitating factors showed a favorable prognostic significance. Ascites, hyperbilirubinemia, poor nutritional state, and hypoprothrombinemia showed bad prognostic significance. This is the first large-scale investigation on hepatic encephalopathy. It demonstrated a similar effectiveness of the two drugs in grade 1 encephalopathy and provides a basis for drug selection in the current management of the syndrome.

     

Beta-lipoprotein- and LDL-associated serum gamma-glutamyltransferase in patients with coronary atherosclerosis

Elevation of serum gamma-glutamyltransferase (GGT) activity is a risk factor for myocardial infarction and stroke. GGT activity can catalyze the oxidation of low-density lipoprotein (LDL), a process involved in the pathogenesis of atherosclerosis. Serum GGT is partially adsorbed onto circulating LDL, and catalytically active GGT has been found within atherosclerotic plaques, colocalizing with oxidized LDL and foam cells. We investigated the the nature of the LDL-associated GGT, the degree of correlation between total serum GGT levels and beta-lipoprotein (beta-LP)-associated GGT, and whether this association is altered in subjects with coronary artery disease (CAD). LDL-bound GGT showed an entire, amphiphilic heavy chain, but the association was easily lost during LDL purification by affinity chromatography. When the activity of GGT associated with polycation-precipitated beta-lipoproteins was assayed, an identical immunoreactive GGT was found in Western blot, and a statistically significant linear correlation was found between total serum GGT levels and the corresponding beta-LP-bound activities (p<0.0001) in controls and patients with CAD. Nevertheless, subjects with CAD presented a lower ratio of beta-LP-bound GGT to total serum GGT respect to controls (p<0.05) and healthy subjects with elevated serum GGT (p<0.01). In addition, a relative decrease of total serum GGT was observed in CAD subjects of older age as compared to younger ones (p<0.005).     

In-vitro testing of three totally supra-annular bileaflet mechanical valves: hydrodynamics in the Sheffield pulse duplicator

BACKGROUND AND AIM OF THE STUDY: In-vivo echocardiographic studies are limited by several confounding factors and technical pitfalls, and consequently the hypothetical differential hydraulic behavior between different prosthetic heart valves has not been identified. However, for surgeons it is essential to know the functional and geometric characteristics of the prostheses to be used. Herein, the in-vitro performance of two new supra-annular bileaflet prostheses--the Medtronic Advantage Supra and Sorin Bicarbon Overline--was compared with that of the 21-mm St. Jude Medical (SJM) Regent valve. METHODS: Three high-performance, production-quality prostheses, including the sewing-ring cuffs, were tested in the aortic chamber of a Sheffield pulse duplicator. The sizes of the prostheses which fitted the 21-mm valve holder were: 21 mm Advantage Supra Medtronic; 19 mm SJM Regent, and 18 mm Sorin Bicarbon Overline. The tests were carried out at a fixed pulse rate (70 beats/min), and at increasing cardiac output (CO) of 2, 4, 5, and 7 l/min. Forward-flow pressure drop, total regurgitant volume, closing and leakage volumes, effective orifice area (EOA) and stroke work loss (SWL) were recorded while the valve was operated at each CO. RESULTS: The SJM Regent and Sorin Bicarbon Overline valves each showed, at increasing CO, significantly lower mean and peak gradients. The calculated EOA and SWL were significantly better with the SJM Regent and Sorin Bicarbon Overline prostheses. The Medtronic Advantage Supra valve showed comparable results only while performing at 2 l/min CO. With regards to the regurgitant fraction, lowest values were observed with the Medtronic Advantage Supra valve. CONCLUSION: This hydrodynamic evaluation model allowed a comparison to be made of the efficiency of recently commercialized bileaflet prostheses, among which the older SJM Regent and the newer Sorin Bicarbon Overline valves demonstrated the best performances.