Antibacterial properties of an oligo-acyl-lysyl hexamer targeting gram-negative species

Toward developing new tools for fighting resistance to antibiotics, we investigated the antibacterial properties of a new decanoyl-based oligo-acyl-lysyl (OAK) hexamer, aminododecanoyl-lysyl-[aminodecanoyl-lysyl](5) (α(12)-5α(10)). The OAK exhibited preferential activity against Gram-negative bacteria (GNB), as determined using 36 strains, including diverse species, with an MIC(90) of 6.2 μM. The OAK's bactericidal mode of action was associated with rapid membrane depolarization and cell permeabilization, suggesting that the inner membrane was the primary target, whereas the observed binding affinity to lipoteichoic acid suggested that inefficacy against Gram-positive species resulted from a cell wall interaction preventing α(12)-5α(10) from reaching internal targets. Interestingly, perturbation of the inner membrane structure and function was preserved at sub-MIC values. This prompted us to assess the OAK's effect on the proton motive force-dependent efflux pump AcrAB-TolC, implicated in the low sensitivity of GNB to various antibiotics, including erythromycin. We found that under sub-MIC conditions, wild-type Escherichia coli was significantly more sensitive to erythromycin (the MIC dropped by >10-fold), unlike its acr-deletion mutant. Collectively, the data suggest a useful approach for treating GNB infections through overcoming antibiotic efflux.     

Cell contact-dependent acquisition of cellular and viral nonautonomously encoded small RNAs

In some organisms, small RNA pathways can act nonautonomously, with responses spreading from cell to cell. Dedicated intercellular RNA delivery pathways have not yet been characterized in mammals, although secretory compartments have been found to contain RNA. Here we show that, upon cell contact, T cells acquire from B cells small RNAs that can impact the expression of target genes in the recipient T cells. Synthetic microRNA (miRNA) mimetics, viral miRNAs expressed by infected B cells, and endogenous miRNAs could all be transferred into T cells. These mechanisms may allow small RNA-mediated communication between immune cells. The documented transfer of viral miRNAs raises the possible exploitation of these pathways for viral manipulation of the host immune response.     

Probiotics for patients with compensated liver cirrhosis: A double-blind placebo-controlled study

Background

Gut flora is related to the major complications of liver cirrhosis including hepatic encephalopathy, spontaneous bacterial peritonitis, and variceal bleeding. Prior studies have reported a beneficial effect of gut flora modification with probiotic bacteria in patients with minimal hepatic encephalopathy. We aimed to study the effect of probiotics on clinical and laboratory parameters of patients with compensated cirrhosis.

Methods

A double-blind placebo-controlled study that included patients with liver cirrhosis and at least one major complication of cirrhosis in the past, clinical evidence of portal hypertension, or decreased hepatic synthetic function. Participants were randomly assigned to receive probiotic capsules containing Lactobacillus acidophilus, Lactobacillus bulgaricus, Bifidobacterium lactis, and Streptococcus thermophiles or placebo for a period of 6 mo.

Results

A total of 36 patients were available for final analysis (distributed equally between the probiotic and placebo groups). The administration of probiotics was not associated with significant differences in either clinical or laboratory parameters between the two groups. Because the lack of a beneficial effect may be related to the compensated liver disease of patients, we conducted a subanalysis of patients with baseline ammonia levels >50 mmol/L. In this subgroup, the administration of probiotics appeared to significantly reduce the ammonia levels starting after 1 mo of treatment. However, this effect diminished and lost its significance following comparison to the placebo group.

Conclusions

Our study did not show a significant beneficial effect of probiotic supplementation in patients with compensated liver cirrhosis. Nevertheless, it points toward a possible positive effect of probiotics in patients with above normal baseline ammonia levels. This issue requires further investigation in larger cohorts.     

Inhibition of lactogenic activities of ovine prolactin and human growth hormone (hGH) by a novel form of a modified recombinant hGH

A recombinant analog of human GH (hGH) lacking 13 amino acids at the amino-terminus (Met14hGH) inhibited the hGH- or ovine PRL (oPRL)-stimulated proliferation of Nb2 lymphoma cells and bovine PRL-stimulated fat synthesis and alpha-lactalbumin secretion in explants from bovine lactating mammary gland. The inhibition was competitive in nature, and in Nb2 cells could be abolished by an excess of hGH or oPRL. Inhibition of oPRL-stimulated proliferation of Nb2 cells by Met14hGH could also be specifically abolished by anti-hGH monoclonal antibodies. Met14hGH had no growth-stimulating activity in Nb2 cells and was not cytotoxic. It also did not affect glucose uptake by the mammary gland explants. Met14hGH competed with [125I]hGH for binding to intact Nb2 cells, IM-9 lymphocytes, solubilized microsomal fraction from lactating bovine mammary gland, and microsomal fraction from the liver of female virgin rats, but its affinity for those receptors was 2 orders of magnitude lower than the affinity of hGH. Since Met14hGH used in most experiments contained about 25% impurities and degradation products, a small amount of it was further purified by immunoaffinity chromatography. Two purified fractions, one consisting of a single 20K protein and the other accompanied by a small amount of 25K protein, were obtained. Both fractions exhibited increased inhibition of hGH- or oPRL-stimulated proliferation of Nb2 cells, thus indicating that the inhibitory activity results from the intact Met14hGH molecule. To the best of our knowledge, this is the first report describing the inhibition of lactogenic hormone activities by a modified hGH.     

Effect of nitric oxide and nitroxide SOD-mimic on the recovery of isolated rat heart following ischemia and reperfusion

Nitric oxide synthesized from L-arginine in cells has important salutary physiological roles, but can also exert deleterious effects. Nitric oxide (NO) can ameliorate post-ischemic reperfusion myocardial injury, yet formation from NO and O(2)z*(-) of peroxynitrite and its downstream toxic products, such as *OH, *NO(2) and CO(3)*(-), can ultimately exacerbate reperfusion damage. Nitroxide stable radicals, such as 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TPL), unlike SOD, readily penetrate cells and catalytically remove intracellular O(2)*(-). Hence, nitroxides by virtue of catalytic removal of O(2)*(-) would be expected to diminish the adverse effect of NO and lower post-ischemic reperfusion cardiac damage. We show that post-ischemic recovery of hemodynamic functions of isolated perfused rat hearts treated with L-arginine or TPL alone did not differ from that of the control hearts. However, the recovery of hearts treated with the combined regimen of L-arginine and TPL was significantly improved, e.g. the Work Index=(left ventricular developed pressure x heart rate) recovered to 92+/-1.6% (L-arginine and TPL) vs. 59.4+/-5.4% (Control), 60+/-2.9% (L-arginine) and 53.3+/-4.3% (TPL) of the pre-ischemic value; mean+/-SEM, N=10, P<0.001. The enhanced recovery of hemodynamic function of hearts treated with L-arginine and TPL was accompanied by an increased recovery of oxygen consumption during the reperfusion. The combined regimen of L-arginine and TPL reduces the negative effects of NO by either inhibiting the production of ONOO(-) or through reaction with CO(3)z.rad;(-) and *NO(2) radicals formed during the decomposition of peroxynitrite in the presence of bicarbonate, thus promoting cardioprotection following post-ischemic reperfusion.     

Hydroxyapatite-electroplated cp-titanium implant and its bone integration potentiality: an in vivo study

PURPOSE: This study was designed to precipitate hydroxyapatite coating on the surface of commercially pure titanium (cpTi) implants using an electroplating technique. After characterization of the hydroxyapatite coating, the bone-implant interface and bone integration of both cpTi and hydroxyapatite-coated implants were assessed. MATERIALS: Twenty implants were divided equally into 2 main groups (n = 10). Ten cpTi implants were utilized as received, while the others were hydroxyapatite coated and then sterilized. The implants of both groups were inserted in the tibiae of New Zealand rabbits. The purity and crystallinity of the hydroxyapatite coat were characterized using x-ray diffraction. A scanning electron microscope examined the grain morphology. Profilometer evaluated the surface texture before and after sterilization. Histological examination using a scanning electron microscope was performed to qualify osseointegration of the regenerated bone and measure the gap distance at the bone-implant interface. RESULTS: Pure crystalline hydroxyapatite precipitate of thickness (range 69-78 microm) and rough surface (2.7 +/- 0.2 microm) compared to smooth cpTi (1.3 +/- 0.5 microm). The gamma-radiation sterilization resulted in finer grains and insignificant smoother surface. Histological examination of the cpTi implant exhibited less bone regeneration with few and less dense bone trabeculae, and gap distance was significantly high (1.29 +/- 0.51 microm). Meanwhile, the hydroxyapatite-coated implant showed a recognizable amount of bone regeneration with more and denser bone trabeculae, and gap distance ranged from 0 to 1.32 microm. CONCLUSIONS: The employed technique provided a thin and uniform pure crystalline hydroxyapatite coating. The characterization of the precipitated film is promising for clinically successful long-term bone fixation.     

Oral variant of acantholytic squamous cell carcinoma—Histochemical and immunohistochemical features

Acantholytic squamous cell carcinoma (ASCC) is an uncommon variant of squamous cell carcinoma (SCC). It is characterized by a combination of typical SCC and pseudoglandular structures, dyskeratotic cells and prominent acantholysis.The purpose of this study was to analyze the histochemical and immunohistochemical characteristics of the intraoral variant of ASCC. Cases of intraoral ASCC were retrieved from the English language literature. Four new cases from our files were added.In total, 35 cases were included and analyzed in this study. The mean age of the patients was 61.5 + 13 years (age range 38–92 years), with a male-to-female ratio of 1.7:1. According to the available data, histochemical and immunohistochemical stains for mucins were found to be consistently negative. E- cadherin, a marker of adherens junctions, was usually reported to be expressed in areas of “typical” (non acantholytic) SCC, but reduced in the acantholytic areas. We examined for the first time the expression of claudin 1, a marker of tight junctions, and found it to be reduced in the acantholytic areas, similar to E-cadherin. Several cases of oral ASCC also expressed vimentin and cytokeratin (CK) 19, markers associated with epithelial-mesenchymal transition. A wide range of non-epithelial markers yielded negative immunoreactions.In conclusion, ASCC is an uncommon variant of squamous cell carcinoma. The acantholytic process appears to involve reduced expression of molecular components of both adherens junctions and tight junctions. These findings could suggest a relation to the epithelial mesenchymal transition process and therefore further studies are needed in order to establish such a link and the subsequent possible impact on the clinical outcome of the patients.