Limosilactobacillus reuteri peptidoglycan alleviates aflatoxin B1-induced toxicity through adsorbing toxins and improving growth, antioxidant status, immunity and liver pathological changes in chicks.

1. The objective of this study was to investigate the protective effects of a peptidoglycan produced by Limosilactobacillus reuteri against aflatoxin B1 (AFB1) induced toxicity in vitro and in vivo in broiler chicks.2. Toxin adsorption experiments were carried out firstly in vitro. These experiments indicated that the absorption efficiency of the peptidoglycan for AFB1 was 64.3-75.9%.3. In the in vivo experiments, Hy-Line Brown chicks were fed a diet containing AFB1 at 71.43 µg/kg with and without peptidoglycan supplementation at concentrations of 100, 200, or 300 g/kg feed from 0-42 d of age.4. The peptidoglycan supplementation in AFB1-contaminated diets resulted in significant improvements in terms of average daily gain, feed intake, feed conversion ratio, white blood cell count, haemoglobin content, glutathione peroxidase activity, immunoglobulin (Ig) A, IgG, IgM and Newcastle disease virus antibody titres (p < 0.05) and diminished liver steatosis.5. In conclusion, peptidoglycan supplementation alleviated AFB1-induced toxicity through adsorbing toxins and improving growth performance, antioxidant ability, immunity and liver pathological changes in chicks. The optimal supplemental dose was 200 mg/kg in feed.

Engineered phage enzymes against drug-resistant pathogens: a review on advances and applications.

In recent decades, the expansion of multi and extensively drug-resistant (MDR and XDR) bacteria has reached an alarming rate, causing serious health concerns. Infections caused by drug-resistant bacteria have been associated with morbidity and mortality, making tackling bacterial resistance an urgent and unmet challenge that needs to be addressed properly. Endolysins are phage-encoded enzymes that can specifically degrade the bacterial cell wall and lead to bacterial death. There is remarkable evidence that corroborates the unique ability of endolysins to rapidly digest the peptidoglycan particular bonds externally without the assistance of phage. Thus, their modulation in therapeutic approaches has opened new options for therapeutic applications in the fight against bacterial infections in the human and veterinary sectors, as well as within the agricultural and biotechnology areas. The use of genetically engineered phage enzymes (EPE) promises to generate endolysin variants with unique properties for prophylactic and therapeutic applications. These approaches have gained momentum to accelerate basic as well as translational phage research and the potential development of therapeutics in the near future. This review will focus on the novel knowledge into EPE and demonstrate that EPE has far better performance than natural endolysins and phages in dealing with antibiotic-resistant infections. Therefore, it provides essential information for clinical trials involving EPE.

Strategies for Using Muramyl Peptides - Modulators of Innate Immunity of Bacterial Origin - in Medicine.

The spread of infectious diseases is rampant. The emergence of new infections, the irrational use of antibiotics in medicine and their widespread use in agriculture contribute to the emergence of microorganisms that are resistant to antimicrobial drugs. By 2050, mortality from antibiotic-resistant strains of bacteria is projected to increase up to 10 million people per year, which will exceed mortality from cancer. Mutations in bacteria and viruses are occurring faster than new drugs and vaccines are being introduced to the market. In search of effective protection against infections, new strategies and approaches are being developed, one of which is the use of innate immunity activators in combination with etiotropic chemotherapy drugs. Muramyl peptides, which are part of peptidoglycan of cell walls of all known bacteria, regularly formed in the body during the breakdown of microflora and considered to be natural regulators of immunity. Their interaction with intracellular receptors launches a sequence of processes that ultimately leads to the increased expression of genes of MHC molecules, pro-inflammatory mediators, cytokines and their soluble and membrane-associated receptors. As a result, all subpopulations of immunocompetent cells are activated: macrophages and dendritic cells, neutrophils, T-, B- lymphocytes and natural killer cells for an adequate response to foreign or transformed antigens, manifested both in the regulation of the inflammatory response and in providing immunological tolerance. Muramyl peptides take part in the process of hematopoiesis, stimulating production of colony-stimulating factors, which is the basis for their use in the treatment of oncological diseases. In this review we highlight clinical trials of drugs based on muramyl peptides, as well as clinical efficacy of drugs mifamurtide, lycopid, liasten and polimuramil. Such a multifactorial effect of muramyl peptides and a well-known mechanism of activity make them promising drugs in the treatment and preventing of infectious, allergic and oncological diseases, and in the composition of vaccines.

The Role of Alveolar Macrophages in the Improved Protection against Respiratory Syncytial Virus and Pneumococcal Superinfection Induced by the Peptidoglycan of Lactobacillus rhamnosus CRL1505.

The nasal priming with nonviable Lactobacillus rhamnosus CRL1505 (NV1505) or its purified peptidoglycan (PG1505) differentially modulates the respiratory innate immune response in infant mice, improving their resistance to primary respiratory syncytial virus (RSV) infection and secondary pneumococcal pneumonia. In association with the protection against RSV-pneumococcal superinfection, it was found that NV1505 or PG1505 significantly enhance the numbers of CD11c+SiglecF+ alveolar macrophages (AMs) producing interferon (IFN)-ß. In this work, we aimed to further advance in the characterization of the beneficial effects of NV1505 and PG1505 in the context of a respiratory superinfection by evaluating whether their immunomodulatory properties are dependent on AM functions. Macrophage depletion experiments and a detailed study of their production of cytokines and antiviral factors clearly demonstrated the key role of this immune cell population in the improvement of both the reduction of pathogens loads and the protection against lung tissue damage induced by the immunobiotic CRL1505 strain. Studies at basal conditions during primary RSV or S. pneumoniae infections, as well as during secondary pneumococcal pneumonia, brought the following five notable findings regarding the immunomodulatory effects of NV1505 and PG1505: (a) AMs play a key role in the beneficial modulation of the respiratory innate immune response and protection against RSV infection, (b) AMs are necessary for improved protection against primary and secondary pneumococcal pneumonia, (c) the generation of activated/trained AMs would be essential for the enhanced protection against respiratory pathogens, (d) other immune and nonimmune cell populations in the respiratory tract may contribute to the protection against bacterial and viral infections, and (e) the immunomodulatory properties of NV1505 and PG1505 are strain-specific. These findings significantly improve our knowledge about the immunological mechanisms involved in the modulation of respiratory immunity induced by beneficial microbes.

Peptidoglycan Suppresses Phagocytic Activities and Apoptosis of Macrophages in Colonic Mucosa Tissues of Crohn's Disease Patients and In Vitro.

BACKGROUND Rac1 signaling plays a crucial role in controlling macrophage functions in CD. Peptidoglycan triggers several intracellular signaling pathways, including activation of Rac1, to regulate the function of macrophage. Suppressed Rac1 signaling in non-inflamed colonic mucosa of Crohn's disease patients has been shown to correlate with increased innate immunity. MATERIAL AND METHODS We examined the effect of peptidoglycan on Rac1 signaling in macrophages and mucosal tissue samples collected from 10 patients with active Crohn's disease and further investigated the effects of peptidoglycan on apoptosis and phagocytic activities of macrophages in vitro. RESULTS Macrophage infiltration and Rac1 signaling was increased in inflamed mucosal tissues of Crohn's disease patients. Immunoblotting assays revealed that peptidoglycan dose- and time-dependently increased the expression of Rac1-GTP, phosphorylated VAV1, and phosphorylated PAK1in RAW264.7 macrophages, which, however, was attenuated by 6-thioguanine. Peptidoglycan also dose-dependently inhibited phagocytic activities of human peripheral blood monocytic cells (PBMCs), which were partially abated by 6-thioguanine or NSC23766. Flow cytometry showed that peptidoglycan (3 µg/mL) decreased the proportion of apoptotic human PBMCs versus controls. The addition of 6-thioguanine or NSC3766 to peptidoglycan led to a sharper rise in the proportion of apoptotic human PBMCs than 6-thioguanine or NSC3766 alone. CONCLUSIONS Our findings suggest that Rac1 signaling is a common molecular target shared by peptidoglycan and immunosuppressive treatment in intestinal macrophages. Inhibiting Rac1 activation may be crucial for optimizing macrophage immunity for treatment of Crohn's disease.

[Role of Rac1 signaling pathway of azathioprine and peptidoglycan in the regulation of monocyte-macrophage apoptosis in Crohn's disease].

Objective: To evaluate the changes of macrophages and expression of Rac1 in the inflammatory site of Crohn's disease, and to investigate the effects of 6-thioguanine (6-TG) and peptidoglycan on apoptosis of human peripheral blood monocyte-macrophage by regulating Rac1 signaling pathway. Methods: Ten patients with Crohn's disease and eight healthy controls diagnosed were enrolled at Department of Gastroenterology and Hepatology, Tianjin Medical University General Hospital from January 2013 to January 2014. The number of macrophages, apoptosis and expression of Rac1 in the inflammation sites and non-inflammation sites of intestinal mucosa were detected in both patients and controls. Peripheral blood mononuclear cells (PBMCs) were sorted by CD14 immunomagnetic beads. The apoptosis of monocytes, expression of Rac1 and related apoptosis signaling molecules were detected in patients treated with peptidoglycan, 6-TG and Rac1 inhibitor NSC23766 and another 15 healthy donors. Results: The number of macrophages and apoptotic cells significantly increased in the inflammatory group of Crohn's disease patients compared with the non-inflammatory group. The expression of PAK1, downstream molecular of Rac1 signaling pathway of macrophages was also significantly higher in the inflammatory group of Crohn's disease patients than that in healthy controls and non-inflammatory group. Compared with control group, anti-apoptotic signals (NF-κB, Bcl-xL and STAT-3) in PBMCs increased in the peptidoglycan group, while slightly decreased in 6-TG group. 6-TG and NSC23766 significantly promoted peptidoglycan-related anti-apoptosis [peptidoglycan group (8.6±3.7)%, peptidoglycan+ 6-TG group (42.0±2.7)%, peptidoglycan+ NSC23766 group (58.5±6.9)%, P<0.05]. Conclusions: Peptidoglycan plays a role in the pathogenesis of Crohn's disease by recruiting macrophages. However, 6-TG inhibits peptidoglycan-induced activation of Rac1 signaling pathway leading to macrophage apoptosis.

Are the immunomodulatory properties of Lactobacillus rhamnosus CRL1505 peptidoglycan common for all Lactobacilli during respiratory infection in malnourished mice?

Previously, we reported that Lactobacillus rhamnosus CRL1505 peptidoglycan (PG05) improves the innate immune response in immunocompromised-malnourished mice after Streptococcus pneumoniae infection. This study extends those previous findings by demonstrating that the dietary recovery of malnourished mice with nasal administration of PG05 improves not only the innate immune response but the respiratory and systemic adaptive humoral response as well. PG05 enhanced the Th2 response, the recovery of B cells, and the concentration and opsonophagocytic activity of anti-pneumococcal antibodies. In addition, by performing comparative studies with the peptidoglycans from lactobacilli of the same species (L. rhamnosus CRL534) or with similar immunomodulatory properties (L. plantarum CRL1506), we demonstrated here that PG05 has unique immunomodulatory properties that cannot be extended to peptidoglycans from other probiotic strains. However, the knowledge of the molecular characteristics of PG05 is indispensable to understand immunomodulatory abilities of L. rhamnosus CRL1505.

CP and CP-PGN protect mice against MRSA infection by inducing M1 macrophages.

Corynebacterium pyruviciproducens (C. pyruviciproducens, CP), as a newly discovered immunomodulator, has been confirmed to have a stronger immunoregulation than Propionibacterium acnes (P. acnes) of the traditional immune adjuvant, by previous experiments with model antigen ovalbumin and sheep red blood cells. Here, it was designed to assess its ability to resist methicillin-resistant Staphylococcus aureus (MRSA), since MRSA as a vital gram positive pathogen is characterized by high morbidity and mortality. In this report, it was indicated that C. pyruviciproducens and its peptidoglycan (CP-PGN) could help to be against bloodstream infection of MRSA with raised survival rate, decreased bacteria load and alleviated systemic inflammation, and these effects of CP-PGN were more pronounced. However, the whole CP was inclined to prevent localized abdominal infection of MRSA from progressing to a systemic infection. And they showed the potential as a therapeutic drug alone or combined with vancomycin. The diversity of capacity of activating macrophages induced by CP and CP-PGN may result in distinct resistance to MRSA in different infection models. Furthermore, both CP and CP-PGN induced M1 macrophages. In conclusion, CP and its PGN could act as promising immune agents to treat and prevent MRSA infection.

Specific oligopeptides in fermented soybean extract inhibit NF-κB-dependent iNOS and cytokine induction by toll-like receptor ligands.

The ethanol extract of fermented soybean from Glycine max (chungkookjang, CHU) has been claimed to have chemopreventive and cytoprotective effects. In the present study, we examined the inhibitory effect of CHU on inducible nitric oxide synthase (iNOS) and cytokine induction by toll-like receptor (TLR) ligands treatment and attempted to identify the responsible active components. Nitric oxide (NO) content and iNOS levels in the media or RAW264.7 cells were measured using the Griess reagent and real-time polymerase chain reaction assays. CHU treatment inhibited NO production and iNOS induction elicited by lipopolysaccharide (LPS, TLR4L) in a concentration-dependent manner. Tumor necrosis factor-α and interleukin-6 productions were also diminished. Peptidoglycans (TLR2/6L) and CpG-oligodeoxynucleotides (TLR9L) from CHU inhibited iNOS induction, but not poly I:C (TLR3L) or loxoribine (TLF7L). The anti-inflammatory effect resulted from the inhibition of nuclear factor-kappa B (NF-κB) through the inhibition of inhibitory-κB degradation. Of the representative components in CHU, specific oligopeptides (AFPG and GVAWWMY) had the ability to inhibit iNOS induction by LPS, whereas others failed to do so. Daidzein, an isoflavone used for comparative purposes, was active at a relatively higher concentration. In an animal model, oral administration of CHU to rats significantly diminished carrageenan-induced paw edema and iNOS induction. Our results demonstrate that CHU has anti-inflammatory effects against TLR ligands by inhibiting NF-κB activation, which may result from specific oligopeptide components in CHU. Since CHU is orally effective, dietary applications of CHU and/or the identified oligopeptides may be of use in the prevention of inflammatory diseases.

The Staphylococcus aureus peptidoglycan protects mice against the pathogen and eradicates experimentally induced infection.

Staphylococcus aureus, in spite of antibiotics, is still a major human pathogen causing a wide range of infections. The present study describes the new vaccine A170PG, a peptidoglycan-based vaccine. In a mouse model of infection, A170PG protects mice against a lethal dose of S. aureus. Protection lasts at least 40 weeks and correlates with increased survival and reduced colonization. Protection extends into drug-resistant (MRSA or VISA) and genetically diverse clinical strains. The vaccine is effective when administered - in a single dose and without adjuvant - by the intramuscular, intravenous or the aerosol routes and induces active as well as passive immunization. Of note, A170PG also displays therapeutic activity, eradicating staphylococci, even when infection is systemic. Sustained antibacterial activity and induction of a strong and rapid anti-inflammatory response are the mechanisms conferring therapeutic efficacy to A170PG.

Collagen-binding peptidoglycans inhibit MMP mediated collagen degradation and reduce dermal scarring.

Scarring of the skin is a large unmet clinical problem that is of high patient concern and impact. Wound healing is complex and involves numerous pathways that are highly orchestrated, leaving the skin sealed, but with abnormal organization and composition of tissue components, namely collagen and proteoglycans, that are then remodeled over time. To improve healing and reduce or eliminate scarring, more rapid restoration of healthy tissue composition and organization offers a unique approach for development of new therapeutics. A synthetic collagen-binding peptidoglycan has been developed that inhibits matrix metalloproteinase-1 and 13 (MMP-1 and MMP-13) mediated collagen degradation. We investigated the synthetic peptidoglycan in a rat incisional model in which a single dose was delivered in a hyaluronic acid (HA) vehicle at the time of surgery prior to wound closure. The peptidoglycan treatment resulted in a significant reduction in scar tissue at 21 days as measured by histology and visual analysis. Improved collagen architecture of the treated wounds was demonstrated by increased tensile strength and transmission electron microscopy (TEM) analysis of collagen fibril diameters compared to untreated and HA controls. The peptidoglycan's mechanism of action includes masking existing collagen and inhibiting MMP-mediated collagen degradation while modulating collagen organization. The peptidoglycan can be synthesized at low cost with unique design control, and together with demonstrated preclinical efficacy in reducing scarring, warrants further investigation for dermal wound healing.

Anti-inflammatory capacity of selected lactobacilli in experimental colitis is driven by NOD2-mediated recognition of a specific peptidoglycan-derived muropeptide.

BACKGROUND AND AIMS: Inflammatory bowel disease (IBD) has been linked to a loss of tolerance towards the resident microflora. Therapeutic use of probiotics is known to be strain specific, but precise mechanisms remain unclear. The role of NOD2 signalling and the protective effect of Lactobacillus peptidoglycan (PGN) and derived muropeptides in experimental colitis were evaluated. METHODS: The anti-inflammatory capacity of lactobacilli and derived bacterial compounds was evaluated using the 2,4,6-trinitrobenzene sulfonic acid (TNBS) colitis model. The role of NOD2, MyD88 and interleukin 10 (IL-10) in this protection was studied using Nod2(-/-), MyD88(-/-) and Il10-deficient mice, while induction of regulatory dendritic cells (DCs) was monitored through the expansion of CD103(+) DCs in mesenteric lymph nodes or after adoptive transfer of bone marrow-derived DCs. The development of regulatory T cells was investigated by following the expansion of CD4(+)FoxP3(+) cells. High-performance liquid chromatography and mass spectrometry were used to analyse the PGN structural differences. RESULTS: The protective capacity of strain Lactobacillus salivarius Ls33 was correlated with a local IL-10 production and was abolished in Nod2-deficient mice. PGN purified from Ls33 rescued mice from colitis in an IL-10-dependent manner and favoured the development of CD103(+) DCs and CD4(+)Foxp3(+) regulatory T cells. In vitro Ls33 PGN induced IL-10-producing DCs able to achieve in vivo protection after adoptive transfer in a NOD2-dependent way. This protection was also correlated with an upregulation of the indoleamine 2,3-dioxygenase immunosuppressive pathway. The protective capacity was not obtained with PGN purified from a non-anti-inflammatory strain. Structural analysis of PGNs highlighted in Ls33 the presence of an additional muropeptide, M-tri-Lys. The synthesised ligand protected mice from colitis in a NOD2-dependent but MyD88-independent manner. CONCLUSIONS: The results indicated that PGN and derived muropeptides are active compounds in probiotic functionality and might represent a useful therapeutic strategy in IBD.

TLR2 ligands attenuate cardiac dysfunction in polymicrobial sepsis via a phosphoinositide 3-kinase-dependent mechanism.

Myocardial dysfunction is a major consequence of septic shock and contributes to the high mortality of sepsis. In the present study, we examined the effect of Toll-like receptor 2 (TLR2) ligands, peptidoglycan (PGN), and Pam3CSK4 (Pam3) on cardiac function in cecal ligation and puncture (CLP)-induced sepsis in mice. We also investigated whether the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway is involved in the effect of TLR2 ligands on cardiac function in CLP mice. PGN was administered to C57B6/L mice 1 h before the induction of CLP. Sham surgically operated mice served as a control. Cardiac function indexes (rate of change in left ventricular pressure, stroke work, cardiac output, and ejection fraction) were examined by a microconductance pressure catheter. Cardiac function was significantly decreased 6 h after CLP-induced sepsis compared with sham-operated control. In contrast, PGN administration attenuated CLP-induced cardiac dysfunction. Importantly, the therapeutic treatment with Pam3 1 h after CLP also significantly attenuated cardiac dysfunction in CLP mice. However, the beneficial effect of TLR2 ligands on cardiac dysfunction in CLP-mice was abolished in TLR2-deficient mice. PGN administration significantly increased the levels of phospho-Akt and phospho-GSK-3beta in the myocardium compared with the levels in untreated CLP mice. PI3K inhibition abolished the PGN-induced attenuation of cardiac dysfunction in CLP mice. In conclusion, these data demonstrate that the administration of TLR2 ligands, PGN, or Pam3 attenuates cardiac dysfunction in septic mice via a TLR2/PI3K-dependent mechanism. More significantly, Pam3 therapeutic treatment will have a potential clinical relevance.

A component of polysaccharide peptidoglycan complex on Lactobacillus induced an improvement of murine model of inflammatory bowel disease and colitis-associated cancer.

Interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) signals play key roles in the pathogenesis of inflammatory bowel disease (IBD). We previously described that both intact cells and a cell wall-derived polysaccharide-peptidoglycan complex (PSPG) in a strain of lactobacillus [Lactobacillus casei Shirota (LcS)] inhibited IL-6 production in lipopolysaccharide (LPS)-stimulated lamina propria mononuclear cells (LPMCs) isolated from murine IBD. Diets with LcS improve murine IBD by suppression of IL-6 synthesis in LPMCs. Moreover, LcS supplementation with fermented milk ameliorates disease activity in patients with active ulcerative colitis. Here, we focused on the specific roles of PSPG in LcS concerning their anti-inflammatory actions. PSPG derived from LcS, and no other strain of lactobacilli, inhibited IL-6 production in LPS-stimulated murine IBD LPMCs. Purified PSPG-I from LcS inhibited IL-6 synthesis in LPS-stimulated murine IBD LPMCs through the inhibition of nuclear factor-kappaB. The anti-IL-6 action of LcS PSPG was abrogated by masking with monoclonal anti-PSPG-I. Furthermore, PSPG-I-negative L. casei strains (PSPG-I-negative mutant LcS: LC(DeltaPSPG-I), L. casei ATCC 334) did not inhibit IL-6 production. Finally, we confirmed the effects of PSPG-I on LcS in the models of both IBD and colitis-associated cancer (CAC). In the IBD model, ingestion of LcS improved ileitis and inhibited activation of IL-6/STAT3 signaling, while ingestion of the LC(DeltaPSPG-I) strain did not. In the CAC model, treatment with LcS, but not the LC(DeltaPSPG-I) strain, showed tumour-suppressive effects with an inhibition of IL-6 production in the colonic mucosa. These results suggested that a specific polysaccharide component in an L. casei strain plays a crucial role in its anti-inflammatory actions in chronic intestinal inflammatory disorders.

Local instillation of Staphylococcus aureus peptidoglycan at operation prevents wound healing impairment after trauma.

BACKGROUND: Considerable experimental evidence and limited clinical evidence indicate that wound healing is impaired after trauma. Because Staphylococcus aureus peptidoglycan (SaPG) accelerates healing in normal rats and prevents wound healing impairment induced by glucocorticoids, cyclophosphamide, and streptozotocin-diabetes, we hypothesized that SaPG would prevent the impaired wound healing after trauma. METHODS: In each of two experiments, 18 Sprague-Dawley male rats were divided into two groups, nine rats each, paired by weight; one group received unilateral comminuted femoral fracture and wounding (two dorsal skin incisions and six subcutaneous polyvinyl alcohol [PVA] sponges), and the other group was only wounded. The incision and PVA sponges on one side were inoculated at operation with saline (200 microL/incision, 50 microL/sponge) and on the other side with SaPG in saline (860 microg of SaPG per centimeter of incision, 0.5 mg of SaPG per sponge). Rats ate chow and drank tap water ad libitum and were killed 7 days postoperatively. RESULTS: In both experiments, the wound breaking strength (WBS) of saline-inoculated incisions was significantly lower in rats with femoral fracture; histologically, reparative granulation tissue was looser and less prominent. WBS of SaPG-inoculated incisions in rats with and without femoral fracture was significantly higher than that of saline-inoculated incisions and, histologically, reparative tissue was more prevalent, more closely packed, and more mature. WBS of SaPG-inoculated incisions in rats with femoral fracture was similar to that of saline-inoculated incisions in rats without femoral fracture. Reparative tissue hydroxyproline and histologic findings of saline-inoculated PVA sponge reparative tissue were similar in all rats, as were the increases induced by SaPG inoculation. CONCLUSION: Wound breaking strength and histologic findings of skin incisions (impaired in rats with unilateral femoral fracture) are more sensitive to the adverse effects of trauma than accumulation of PVA sponge reparative tissue. A single inoculation of SaPG at operation increased wound incision healing in rats both without and with femoral fracture and notably prevented the impaired healing in rats with femoral fracture.

Anti-tumor activity of squid ink.

The anti-tumor activity of a new type of peptidoglycan isolated from squid ink was shown to have a cure rate of 64% for Meth A tumor from BALB/c mice. The ink delipidated in acetone, which contained the peptidoglycan at 0.1% (w/w), was administered to tumor-transplanted mice so as to examine the anti-tumor activity. One-fifth of the tumor-bearing mice was cured with 3 injections (1 mg/head) of the acetone delipidated squid ink or a prolongation of survival was observed in the treated animals. Heat treatment at 100 degrees C for 10 min did not affect the anti-tumor activity of the delipidated ink, its potentiality being preserved. The acetone-extractable fraction of the ink also brought about a similar cure rate for Meth A tumor. The delipidated ink enhanced the phagocytic activity of macrophages but no direct cytotoxicity was observed for the Meth A tumor cells. Hence it may be said that the anti-tumor activity of the delipidated ink was mainly due to the augmented cellular immunity in vivo.

Antitumor activity of bacteria and bacterial products: enhancement of the tumor-protective effect of bacteria by lipoteichoic acid.

The ability of some microbial agents and/or their products to affect local tumor growth was assessed in the D-12 DA rat ascites tumor model. Various bacteria and bacterial products markedly enhanced tumor resistance when injected i.p. several days before tumor cell challenge. The tumor-protective effect of these compounds was amplified further by lipoteichoic acid (LTA) inoculated i.p. a few days after tumor cell challenge. Under these conditions, the majority of animals did not exhibit progressive tumor growth.

An investigation of the antitumor peptidoglycan fraction from squid ink.

Squid ink, which has little commercial use and is usually discarded, was extracted using a Tris-HCl buffer (pH 6.8). The extract was fractionated using DEAE Sephacel ion-exchange chromatography and Sephacryl S-300 gel filtration to give a peptidoglycan fraction which exhibited strong antitumor activity against Meth-A fibrosarcoma in BALB/c mice following intraperitoneal administration. The fraction was composed of 7.8% peptide, 57% polysaccharide and 30% pigment. The polysaccharide component had a unique structure with equimolar ratios of GlcA, GalNAc and Fuc. Since the fraction has no direct cytotoxic effect on Meth-A cells, inhibition of tumor growth may be due to stimulation of host-mediated responses.