A novel chimeric phage lysin with high in vitro and in vivo bactericidal activity against Streptococcus pneumoniae.

OBJECTIVES: Streptococcus pneumoniae is becoming increasingly antibiotic resistant worldwide and new antimicrobials are urgently needed. Our aim was new chimeric phage endolysins, or lysins, with improved bactericidal activity by swapping the structural components of two pneumococcal phage lysozymes: Cpl-1 (the best lysin tested to date) and Cpl-7S. METHODS: The bactericidal effects of four new chimeric lysins were checked against several bacteria. The purified enzymes were added at different concentrations to resuspended bacteria and viable cells were measured after 1 h. Killing capacity of the most active lysin, Cpl-711, was tested in a mouse bacteraemia model, following mouse survival after injecting different amounts (25-500 µg) of enzyme. The capacity of Cpl-711 to reduce pneumococcal biofilm formation was also studied. RESULTS: The chimera Cpl-711 substantially improved the killing activity of the parental phage lysozymes, Cpl-1 and Cpl-7S, against pneumococcal bacteria, including multiresistant strains. Specifically, 5 µg/mL Cpl-711 killed ≥7.5 log of pneumococcal R6 strain. Cpl-711 also reduced pneumococcal biofilm formation and killed 4 log of the bacterial population at 1 µg/mL. Mice challenged intraperitoneally with D39_IU pneumococcal strain were protected by treatment with a single intraperitoneal injection of Cpl-711 1 h later, resulting in about 50% greater protection than with Cpl-1. CONCLUSIONS: Domain swapping among phage lysins allows the construction of new chimeric enzymes with high bactericidal activity and a different substrate range. Cpl-711, the most powerful endolysin against pneumococci, offers a promising therapeutic perspective for the treatment of multiresistant pneumococcal infections.

Antitussive and immunomodulating activities of instant coffee arabinogalactan-protein.

A low molecular mass arabinogalactan-protein (AGP) composed of galactose and arabinose with a low protein content, isolated from the instant coffee powder of Coffea arabica beans, has been tested on antitussive (in vivo) and immunomodulating (ex vivo) activities. The results of antitussive tests revealed a significant dose dependant cough-suppressive effect of coffee AGP. It was observed 30 or 60 min after AGP administration and its efficacy lasted during the entire experiment course. Immunological tests showed that AGP affected some mediators of immunocompetent cells of immune system as TNF-α, IFN-γ and IL-2 cytokines. It seems that coffee AGP is a good inductor of both pro-inflammatory cytokines TNF-α and IFN-γ, however, less potent in TNF-α induction in comparison with that of ß-D-glucan. Evident induction of TNF-α, IL-2 and IFN-γ cytokines, pro-TH1 polarization supports our conclusion about bio-immunological efficacy of AGP with an emphasis on the cellular immunity.

The effects of an arabinogalactan-protein from the white-skinned sweet potato (Ipomoea batatas L.) on blood glucose in spontaneous diabetic mice.

We examined the effects of an arabinogalactanprotein (WSSP-AGP) from Ipomoea batatas L. on hyperglycemia in db/db mice. An oral glucose tolerance test indicated significantly decreased plasma glucose levels by WSSP-AGP. Additionally, an insulin tolerance test found improvement in insulin sensitivity due to treatment with WSSP-AGP. This suggests that amelioration of insulin resistance by WSSP-AGP causes to lead its hypoglycemic effects.

LysGH15, a novel bacteriophage lysin, protects a murine bacteremia model efficiently against lethal methicillin-resistant Staphylococcus aureus infection.

Phage-coded lysin is an enzyme that destroys the cell walls of bacteria. Phage lysin could be an alternative to conventional antibiotic therapy against pathogens that are resistant to multiple antibiotics. In this study, a novel staphylococcal phage, GH15, was isolated, and the endogenous lytic enzyme (LysGH15) was expressed and purified. The lysin LysGH15 displayed a broad lytic spectrum; in vitro treatment killed a number of Staphylococcus aureus strains rapidly and completely, including methicillin-resistant S. aureus (MRSA). In animal experiments, a single intraperitoneal injection of LysGH15 (50 µg) administered 1 h after MRSA injections at double the minimum lethal dose was sufficient to protect mice (P < 0.01). Bacteremia in unprotected mice reached colony counts of about 10(7) CFU/ml within 3.5 h after challenge, whereas the mean colony count in lysin-protected mice was less than 10(4) CFU/ml (and ultimately became undetectable). These results indicate that LysGH15 can kill S. aureus in vitro and can protect mice efficiently from bacteremia in vivo. The phage lysin LysGH15 might be an alternative treatment strategy for infections caused by MRSA.

A novel chimeric lysin shows superiority to mupirocin for skin decolonization of methicillin-resistant and -sensitive Staphylococcus aureus strains.

Staphylococcus aureus is a major human pathogen responsible for a number of serious and sometimes fatal infections. One of its reservoirs on the human body is the skin, which is known to be a source of invasive infection. The potential for an engineered staphylococcus-specific phage lysin (ClyS) to be used for topical decolonization is presented. We formulated ClyS into an ointment and applied it to a mouse model of skin colonization/infection with S. aureus. Unlike the standard topical antibacterial agent mupirocin, ClyS eradicated a significantly greater number of methicillin-susceptible S. aureus (MSSA) and -resistant S. aureus (MRSA) bacteria: a 3-log reduction with ClyS as opposed to a 2-log reduction with mupirocin in our model. The use of ClyS also demonstrated a decreased potential for the development of resistance by MRSA and MSSA organisms compared to that from the use of mupirocin in vitro. Because antibodies may affect enzyme function, we tested antibodies developed after repeated ClyS exposure for their effect on ClyS killing ability. Our results showed no inhibition of ClyS activity at various antibody titers. These data demonstrate the potential of developing ClyS as a novel class of topical antimicrobial agents specific to staphylococcus.

Structural characterization and hypoglycemic effects of arabinogalactan-protein from the tuberous cortex of the white-skinned sweet potato (Ipomoea batatas L.).

An arabinogalactan-protein (WSSP-AGP) was isolated from the tuberous cortex of the white-skinned sweet potato (WSSP; Ipomoea batatas L.). It consists of 95% (w/w) carbohydrate and 5% (w/w) protein with high contents of hydroxyproline, alanine, and serine. Its sugar composition is α-L-Rha:α-L-Ara:ß-D-Gal:ß-D-GlcA in a molar ratio of 1.0:4.1:7.6:1.3. Its weight-average molecular weight was estimated to be 126,800 g/mol by high-performance size exclusion chromatography coupled with multiangle laser light scattering. Structural analysis indicated that WSSP-AGP is a (1→3)-ß-D-galactan highly branched at O-6 with (1→6)-ß-D-galactan, in which the branched chains are substituted at the O-3 position with α-L-Araf-(1→ and α-L-Araf-(1→5)-α-L-Araf-(1→ and at the O-6 position typically with α-L-Rhap-(1→4)-ß-D-GlcAp-(1→ as terminating groups. Continuous administration of WSSP-AGP to KKAy mice significantly lowered fasting plasma glucose levels. This indicates that WSSP-AGP plays an important role in the hypoglycemic effects of WSSP.

Synergy of Nocardia rubra cell wall skeleton and interleukin 2 in the in vivo induction of murine lymphokine-activated killer cell activity.

Combination of an ip injection of Nocardia rubra cell wall skeleton (N-CWS) and 3 daily sc injections of human recombinant interleukin 2 (rIL 2) into C3H/HeN mice resulted not only in a significant increase in the number of peritoneal cells (PC) but also in a potent induction of their lymphokine-activated killer (LAK) activity, compared with results obtained with N-CWS or rIL 2 alone. The augmented LAK activity of PC was mediated by nonadherent, nonphagocytic, Thy-1.2+(-)- and asialo GM1+ cells. Nonadherent PC induced by an ip injection of N-CWS bound more 125I-labeled rIL 2 than did normal, nonadherent PC, and generated high LAK activity when cultured overnight with rIL 2. In contrast, normal, nonadherent PC responded only weakly to the overnight stimulation with rIL 2. The phenotype of N-CWS-induced PC with an elevated IL 2 responsiveness was Thy-1.2+(-)-, Lyt-1.1-, Lyt-2.1- and asialo GM1+, suggesting that the N-CWS-stimulated LAK precursors were derived mainly from the NK cell lineage. However, mature T cells may also be involved in this mechanism, because N-CWS failed to augment the IL 2 responsiveness of nonadherent PC in BALB/c nu/nu mice. Treatment of C57BL/6N mice bearing solid Lewis lung carcinoma (3LL) tumors with an intratumoral injection of N-CWS followed by 6 daily sc injections of rIL 2 resulted in the apparent suppression of tumor growth, while N-CWS or rIL 2 alone produced no such suppression. These results suggest that N-CWS augments the antitumor effect of rIL 2 by accumulating LAK precursors and elevating their responsiveness to rIL 2 at the injection site.

Current status of melanoma treatment with interferon, cytokines and other biologic response modifiers in Japan.

This paper introduces the current status of melanoma treatment with various biologic response modifiers (BRMs) in Japan, with an emphasis on the clinical results of Interferon therapies. The authors also refer briefly to the current situation of interleukin-2 (IL-2) and tumor necrosis factor (TNF) in Japan. Many BRMs have been used in treatment of melanoma, e.g., IFN, IL-2, TNFs, BCG, MY-1 (DNA extracted from BCG), WPG (CWs of Bifidobacterium infantis, ATCC 15697), OK-432 (Picibanil, Streptococcus pyogenes preparation), bestatin, and forphenicinol. Some of these have completed clinical trials, while others are still undergoing clinical testing. Among IFN-alpha, beta, and gamma, intralesional administration of natural IFN-beta was found to be more effective than IFN-alpha for metastatic skin melanoma, the survival time of patients being prolonged by the administration of IFN-beta. IFN-gamma appeared to have lower efficacy than IFN-alpha and beta. The frequency of BRM application to melanoma treatment will increase. The authors foresee that combinations with radio- and/or other chemotherapy will be more common than the single use of a BRM, especially in the case of IFN.

Induction of tumoricidal macrophages from bone marrow cells of normal mice or mice bearing a colony-stimulating-factor-producing tumor.

Nonadherent cells of the bone marrow of C3H/HeN mice were incubated for 3 days with the culture supernatant of an L-929 cell line containing macrophage-colony-stimulating factor. Approximately, 70% of the cells became phagocytic, adherent to plastic dishes and positive for nonspecific esterase staining. The adherent cells exhibited a weak tumoricidal activity against syngeneic mammary carcinoma cells, and the cytotoxicity was strongly augmented by the addition of bacterial lipopolysaccharide to the cytotoxicity assay. The cytotoxicity induced by lipopolysaccharide was also shown to be mediated by Thy1.2- and asialo-GM1+ cells, and was abrogated by the addition of carrageenan. Macrophage-colony-stimulating-factor-producing (D66) and nonproducing (A23) variants were separated from the MM48 tumor line in in vitro culture following limiting dilution. There was no difference between these two variants in either the in vitro growth rate or the susceptibility to macrophage-mediated cytotoxicity. C3H/HeN mice inoculated i.p. with D66 survived longer than did those inoculated i.p. with A23. C3H/HeN mice bearing D66 or A23 as an ascitic form were given i.p. injections of Nocardia rubra cell wall skeleton (N-CWS). N-CWS significantly prolonged the survival period of mice bearing D66, whereas it exhibited no apparent antitumor effect on mice bearing A23. The increase in the cell number of D66 in the peritoneal cavity was significantly retarded, compared with that of A23. In contrast, the number of peritoneal macrophages increased more in D66-bearing mice than in A23-bearing mice. The increase in the peritoneal macrophage number was further augmented by an i.p. injection of N-CWS. Peritoneal macrophages of D66-bearing mice exhibited apparent tumoricidal activity against MM48 tumor cells in the presence of lipopolysaccharide, and the cytotoxicity was significantly augmented by i.p. injection of N-CWS. On the other hand, the responsiveness of peritoneal macrophages to lipopolysaccharide was found to be poor in A23-bearing mice and the tumoricidal activity was only weakly augmented by N-CWS. These results strongly suggest that M-CSF plays an important role not only in the maturation of macrophage progenitors but also in the induction and the accumulation of activated macrophages.

Effects of periodic administration of Nocardia rubra cell-wall skeleton on immunoglobulin production and B-cell-stimulatory factor activity in vitro in workers at a poison gas factory.

The former workers at the Okunojima poison gas factory (poison gas workers) are a high-risk group for malignant neoplasms and show abnormalities in cellular immunity. At the same time, poison gas workers often have chronic respiratory diseases, such as chronic bronchitis, and are highly susceptible to respiratory infections. To explore the possibility of immunological cancer prevention, we have periodically administered 200 micrograms Nocardia rubra cell-wall skeleton (N-CWS) to poison gas workers once every 3 months since December 1978. During this period, we noted a significantly lower incidence of influenza among poison gas workers receiving N-CWS than in those not receiving the drug during the influenza epidemic. This finding suggested that the administration of N-CWS enhanced the resistance of these workers to infections. Therefore, periodical administration of N-CWS to poison gas workers was considered to enhance the reduced T-cell function of normalizing antibody production by stimulating the production of B-cell-stimulatory factor (BSF). In the present study, to clarify the mechanism of immunosuppression in the poison gas workers and to examine the effects of continual administration of N-CWS on this condition, we compared the immunoglobulin production and the proliferative and differentiative activities of B-cell-stimulatory factor (BSF) of peripheral blood mononuclear cells (PBMC), in poison gas workers treated or not treated with N-CWS. Comparisons were also made with age-matched healthy controls. In the untreated poison gas workers, immunoglobulin and BSF production of PBMC were reduced as compared with the control group. On the other hand, in the poison gas workers receiving N-CWS, immunoglobulin and BSF production of PBMC were restored nearly to the control level. These results show that in vitro antibody production in the poison gas workers was reduced and that a reduction in BSF production of T cells was one of its causes.

Protective effect of Nocardia rubra cell wall skeleton on experimental infection in normal and immunosuppressed mice.

The protective effect of Nocardia rubra cell wall skeleton (N-CWS) on experimental infections was investigated in normal and immunosuppressed mice. Pretreatment with N-CWS provided protection against acute systemic infections due to Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Serratia marcescens and Listeria monocytogenes in normal mice. N-CWS administered before or after challenge also showed protective activity against Herpes simplex virus infection in normal mice. N-CWS was the most effective against extracellular parasitic Pseudomonas aeruginosa infection when administered 1 day before challenge, but displayed the most potent protective activity against infection with Listeria monocytogenes, a facultative intracellular parasite, when administered 7 to 14 days before challenge. In mice with subcutaneous Pseudomonas aeruginosa infection, N-CWS suppressed abscess formation and decreased the viable cell count in the resultant abscess foci when administered either intraperitoneally or subcutaneously to the infected site. In addition, treatment with N-CWS markedly restored host defense ability against pseudomonal infection in mice immunosuppressed with cyclophosphamide, hydrocortisone and X-ray irradiation.

[Reduction of post-heparin lipoprotein lipase activity by acidotic blood pH]. / Aktivitätsverminderung der post-Heparin-Lipoproteinlipase bei azidotischem Blut-pH.

Diseases associated with acidotic blood-pH, such as chronic renal disease, diabetes mellitus or chronic alcoholism, show a marked impairment of lipoprotein lipase. Therefore we influenced blood-pH in 3 healthy subjects by infusions to get alkalotic, neutral and acidotic blood-pH on three days in series. On each day blood-pH from capillary blood and post-heparin lipoprotein lipase from fasting plasma was determined. In comparison to neutral blood-pH in vivo, alkalosis did not influence lipoprotein lipase. In contrast, during artificial acidosis, lipoprotein lipase was impaired significantly (p less than 0.01). Therefore, it seems, that acidosis inhibits lipoprotein lipase in vivo.

Stimulation of non-specific anti-tumour resistance in the mouse using cell wall preparations from four BCG substrains.

The comparative anti-tumour activity of cell walls, deproteinized cell walls and cell wall skeleton (CWS) isolated from four BCG substrains (Canadian, Russian, Glaxo and Swedish) was determined against Ehrlich ascitic carcinoma in CF-1 mice and against L1210 lymphoid leukaemia in B6D2F1/J mice. In Ehrlich carcinoma, the protocol of injection was shown to be of critical importance, since high and low levels of protection and even facilitation of tumour growth were observed according to the protocol used. The oil emulsion used as the vehicle for injection of the fractions (control groups) induced facilitation of tumour growth with some protocols. The highest levels of protection were observed when treatment was performed on day -6 for a single i. p. injection or on days -14 and 0 for two injections. Using the former protocol of immunization, the highest level of protection was detected with cell walls and deproteinized cell walls. CWS isolated from the Canadian and Glaxo substrains were found to be inactive, whereas those isolated from the Swedish and Russian substrains induced significant levels of protection. In L1210 leukaemia, very few preparations induced significant protection. CWS preparations did not induce protection. Canadian and Russian deproteinized cell walls and Canadian cell walls (100 and 250 micrograms, respectively) induced some protection. The use of different protocols of injection did not increase the anti-tumour activity of the Canadian cell walls. The most active preparation in both tumour systems was that of Canadian deproteinized cell walls.

[Effects of administration of gastric mucoprotein and vitamin B 12 on rats fed Hauk's and Osers standard diet and Wynn's cariogenic diet]. / Effetti della somministrazione di mucoproteina gastrica e vit. B12 nei ratti alimentati con dieta standard di Hauk e Oser e con dieta cariogena di Wynn (emory)

An increase in dental decay of 48.89% over controls fed with a cariogenic diet was noted when gastric mucoprotein and vitamin B12 were administered to rats receiving a standard diet.

H-2-linked genetic control of immune responsiveness to ovalbumin and ovomucoid.

Immune responsiveness of inbred mice to low doses of ovalbumin or ovomucoid is under control of single dominant genes closely linked to alleles of the H-2 locus. High responsiveness to ovomucoid is linked with the H-2(a) and H-2(k) alleles, and to ovalbumin with the H-2(b), H-2(d), and H-2(q) alleles.

[Protidogram and serum agglutinin data in rabbits subjected to vaccination and gastric mucoprotein administration]. / Comportamento del protidogramma e delle globuline sieriche in conigli sottoposti a trattamento con mucoproteina gastrica e vaccino.

A statistical assessment was made of the results obtained in a study of the behaviour of the protidogram and serum agglutinins in rabbits subjected to Salmonella typhi murium vaccination and 25 and 50 mg/kg per day gastric mucoprotein. Albumin values were decreased in animals both vaccinated and treated, moderately increased in those that received mucoprotein only. Globulins increased in both groups. Antibody increases were most marked in animals vaccinated and treated with the larger mucoprotein dose.