Immunocycte stimulation in vitro by nontoxic bacterial lipopolysaccharide derivatives.

Intact lipopolysaccharides (LPS), considered nonspecific enhancers of B cell responses, as well as nontoxic derivatives from Serratia marcescens LPS, were studied with regard to their ability to stimulate in vitro immune responses to a T-dependent antigen, sheep erythrocytes. Intact LPS, at a dose of 10 to 50 microgram, consistently enhanced the in vitro anti-SRBC immune response by normal splenocytes. The LPS also increased the background PFC response to SRBC in nonimmunized cultures. A chemically detoxified preparation derived from LPS (Mex B) had no stimulatory activity in vitro. A completely nontoxic, relatively small m.w., polysaccharide-rich preparation (PS), free of detectable lipid and protein, was stimulatory in vitro and at a dose of 10 microgram resulted in a 40 to 70% enhancement of the anti-SRBC response. The PS also stimulated an enhanced background response to SRBC as well as several other RBC species in nonimmunized cultures. PS had no mitogenic effect in vitro since addition of this bacterial derivative failed to stimulate thymidine incorporation into mouse splenocytes, as occurred with the intact LPS. The use of nontoxic preparations from gram-negative bacterial LPS for dissecting the stimulatory vs antigenic properties of bacterial products provides a model system for determining the role of a mitogenic stimulus in B cell activation.     

Combined immunostimulation in the prevention of tumor take in mice using endotoxins,their derivatives,and other immune adjuvants

Summary In this study a variety of immunostimulatory agents was tested alone or in combination with other agents to measure their ability to enhance nonspecific resistance to challenge with the TA3-Ha transplantable murine ascites tumor. Lipopolysaccharides (LPS) were effective alone in protecting mice from tumor. Their lipid-free, nontoxic, polysaccharide-rich hydrolytic products (PS) showed a much reduced, but still significant effect in anti-TA3-Ha resistance induction. Other agents that stimulated tumor resistance included nontoxic native hapten and trehalose-6,6-dimycolate (P3) of Ribi, synthetic glycolipids, cord factor, and killed BCG preparations. Simultaneous pretreatment with a combination of any of these agents and either LPS or PS resulted in a significantly higher level of tumor resistance. Administration of LPS, PS, or native hapten to mice that had been previously infected with viable BCG resulted in the strongest antitumor effect. These studies demonstrate that nonspecific resistance can be enhanced in an additive or synergistic manner by using combinations of agents, which presumably stimulate different cell populations of the host's immune system.     

Phosphatidylserine regulates the maturation of human dendritic cells

Phosphatidylserine (PS), which is exposed on the surface of apoptotic cells, has been implicated in immune regulation. However, the effects of PS on the maturation and function of dendritic cells (DCs), which play a central role in both immune activation and regulation, have not been described. Large unilamellar liposomes containing PS or phosphatidylcholine were used to model the plasma membrane phospholipid composition of apoptotic and live cells, respectively. PS liposomes inhibited the up-regulation of HLA-ABC, HLA-DR, CD80, CD86, CD40, and CD83, as well as the production of IL-12p70 by human DCs in response to LPS. PS did not affect DC viability directly but predisposed DCs to apoptosis in response to LPS. DCs exposed to PS had diminished capacity to stimulate allogeneic T cell proliferation and to activate IFN-gamma-producing CD4(+) T cells. Exogenous IL-12 restored IFN-gamma production by CD4(+) T cells. Furthermore, activated CTLs proliferated poorly to cognate Ag presented by DCs exposed to PS. Our findings suggest that PS exposure provides a sufficient signal to inhibit DC maturation and to modulate adaptive immune responses.     

Adult female crickets, Gryllus texensis, maintain reproductive output after repeated immune challenges

Abstract.  Both immunity and reproduction are thought to be energetically costly and therefore likely to make trade-offs with one another. To assess whether increasing immune system activity results in a decline in egg production, the immune system in the cricket Gryllus texensis is activated over a period of 12 days with regular injections of lipopolysaccharide (LPS) derived from Serratia marcescens , and the number of eggs laid during this time counted. Egg quality is also assessed by measuring total protein of eggs laid, fertilization and hatching success, and the weight of individual eggs laid after the series of injections. Indirect evidence suggests that LPS induces an immune response in G. texensis . However, the number of eggs produced is not affected. There is also no effect of repeated LPS injections on female weight, egg protein content, or fertilization and hatching success. Taken together, these results suggest that with food and water provided ad libitum , females can protect many aspects of fitness in the face of increased immune system activity. However, there is some evidence to suggest that large (100 μg) doses of LPS lead to reduced female longevity, and also in egg weight that could affect offspring success. Although the possibility exists that the decline in lifespan and egg weight after high-dose injections reflects a trade-off between reproduction and immune investment, another possibility is that these doses yield nonspecific effects, or that the high-dose induces an overwhelming immune response that leads to self-damage.     

The characteristics of the O-specific humoral immune response of mice vaccinated with Salmonella choleraesuis antigens]

The work deals with the results of the comparative enzyme immunoassay (EIA) of serum samples taken from (CBA X C57BL/6) F1 mice immunized with O-specific polysaccharides, O-antigens (O-Ag) obtained by Boivin's method and antigenic preparations isolated with hydroxylamine (HA) from S. choleraesuis and S. typhimurium. O-Ag and lipopolysaccharide (LPS) of the corresponding bacterial species were used as antigens for the sensitization of polystyrene plates. The primary and secondary humoral immune response was studied by means of EIA. As revealed in this investigation, the immunization of mice with HA-isolated antigenic preparations and O-Ag, obtained from S. typhimurium, in a single injection (in doses of 1-100 micrograms) led to the development of weak specific immune response to O-Ag. Response to LPS was absent. After the second immunization of the animals pronounced immune response to O-Ag and LPS was observed. It developed as a response of both IgM and IgG type. The immunization of mice, made in a single injection, with HA-isolated antigenic preparations and O-Ag, obtained from S. choleraesuis, did not lead to the development of O-specific immune response. After the immunization of mice with these antigens in two injections sharply pronounced nonspecific activity of IgM and IgG serum antibodies with respect to O-Ag and LPS of homologous and heterologous bacterial species was noted in EIA. Neither S. typhimurium O-polysaccharide, nor S. choleraesuis O-polysaccharide did not induce O-specific immune response even after the second immunization.     

Transgenic mice expressing a fully nontoxic diphtheria toxin mutant, not CRM197 mutant, acquire immune tolerance against diphtheria toxin

We previously developed a method termed "toxin receptor-mediated cell knockout" (TRECK). By the TRECK method, a single or repeated shot(s) of diphtheria toxin (DT) conditionally ablates a specific cell population from transgenic mice expressing the DT receptor transgene under the control of a cell type-specific promoter. In some cases of TRECK, frequent and high-dose administration of DT is required, raising the concern that these frequent injections of DT could cause production of anti-DT antibody, which would neutralize further DT administration. To solve this problem, we aimed to generate transgenic mice genetically expressing a nontoxic DT mutant, with the expectation that they may naturally acquire immune tolerance to DT. Unexpectedly, the G52E DT mutant, which is well known as the nontoxic DT variant cross reacting material 197 (CRM197), exhibited cytotoxicity in yeast and mammalian cells. Cytotoxicity of CRM197 was abrogated in cells mutated for elongation factor 2 (EF-2), indicating that CRM197 exerts its toxic effects through EF-2, similar to wild-type DT. On the other hand, the K51E/E148K DT mutant exhibited no detectable cytotoxicity. This led us to successfully obtain DT gene transgenic mice, which exhibited no histological abnormalities, and indeed acquired immune tolerance to DT.     

Enhanced antibody response in retrovirus-infected mice treated with endotoxin or nontoxic polysaccharide derivative

Friend leukemia virus (FLV) is a retrovirus which causes marked suppression of the immune response of genetically susceptible mice. In the present study the depressed antibody response to sheep erythrocytes by spleen cells from FLV-infected mice was partially reversed by injection of either a bacterial endotoxin or a nontoxic polysaccharide derivative directly into infected mice or by addition to spleen cell cultures from these mice immunized in vitro with sheep red blood cells (SRBC). The endotoxin and PS in a dose-related manner markedly increased the antibody responsiveness of the spleen cells to SRBC. Thus these results indicate that the nontoxic polysaccharide derivative has properties equivalent to the toxic endotoxin in enhancing the antibody responsiveness of FLV-suppressed spleen cells to a T-cell-dependent antigen like SRBC.     

Pharmacokinetic modeling of an induction regimen for in vivo combined testing of novel drugs against pediatric acute lymphoblastic leukemia xenografts

Current regimens for induction therapy of pediatric acute lymphoblastic leukemia (ALL), or for re-induction post relapse, use a combination of vincristine (VCR), a glucocorticoid, and L-asparaginase (ASP) with or without an anthracycline. With cure rates now approximately 80%, robust pre-clinical models are necessary to prioritize active new drugs for clinical trials in relapsed/refractory patients, and the ability of these models to predict synergy/antagonism with established therapy is an essential attribute. In this study, we report optimization of an induction-type regimen by combining VCR, dexamethasone (DEX) and ASP (VXL) against ALL xenograft models established from patient biopsies in immune-deficient mice. We demonstrate that the VXL combination was synergistic in vitro against leukemia cell lines as well as in vivo against ALL xenografts. In vivo, VXL treatment caused delays in progression of individual xenografts ranging from 22 to >146 days. The median progression delay of xenografts derived from long-term surviving patients was 2-fold greater than that of xenografts derived from patients who died of their disease. Pharmacokinetic analysis revealed that systemic DEX exposure in mice increased 2-fold when administered in combination with VCR and ASP, consistent with clinical findings, which may contribute to the observed synergy between the 3 drugs. Finally, as proof-of-principle we tested the in vivo efficacy of combining VXL with either the Bcl-2/Bcl-xL/Bcl-w inhibitor, ABT-737, or arsenic trioxide to provide evidence of a robust in vivo platform to prioritize new drugs for clinical trials in children with relapsed/refractory ALL.     

Intranasal immunization of mice with CpG DNA induces strong systemic and mucosal responses that are influenced by other mucosal adjuvants and antigen distribution

BACKGROUND: Synthetic oligodeoxynucleotides (ODN) containing immunostimulatory cytosine-guanine phosphate-linked dinucleotide (CpG) motifs are potent systemic and mucosal adjuvants in mice that have synergistic action with numerous other adjuvants, including alum and cholera toxin (CT). Herein, we evaluate CpG ODN with intranasal (IN) delivery of purified hepatitis B surface antigen (HBsAg), relative to and in combination with CT, Escherichia coli heat labile enterotoxin (LT), the B subunit of CT (CTB), and a nontoxic derivative of LT (LTK63). MATERIALS AND METHODS: BALB/c mice were immunized by IN administration of HBsAg, alone or combined with CT, LT, CTB, or LTK63, and/or CpG ODN, or non-CpG control ODN. In addition, the effect of low-or high-volume administration was assessed, in order to target upper respiratory or entire respiratory tract, respectively. HBsAg-specific systemic (immunoglobulins: IgG, IgG1, IgG2a in plasma) and mucosal (IgA in fecal, lung, vaginal, saliva, and gut samples) humoral responses, as well as cell-mediated immune responses including T-cell proliferation and cytokines (interleukins: IL-4, IL-5; interferon: IFN-gamma) were evaluated. RESULTS: CpG ODN, CT, and LT augmented anti-HBs titers equally, and more so than did CTB or LTK63. CpG ODN acted synergistically with CT and LT, but not CTB or LTK63 to enhance anti-HBs titers. Nevertheless, CpG ODN induced a more Th1-like response for all combinations, compared with the same formulation without CpG. Strength of induced systemic and mucosal immune responses was better with IN delivery of a large volume. A small volume required multiple administrations and higher doses of antigen and adjuvant for equal results. This suggests that delivery of antigen to the lung and/or diges-tive system is superior to delivery to the nasal cavity. CONCLUSIONS: Our results suggest that the synergy between CpG ODN and native toxins (CT, LT) may depend on their enzymatic activity and that the lack of synergy with nontoxic derivatives (LTB, LTK63) arises, since they do not have enzymatic activity. Because both CT and LT are too toxic for use in humans, it is possible that CpG ODN may be combined with bacterial toxin mutants that retain some enzymatic activity to optimize immune augmentation.     

Effects of single or repeated administrations of methamphetamine on immune response in mice.

The present study aimed to clarify the connection between immune responses and the administration frequency of methamphetamine (MAP) in male and female mice. Male and female ddY mice were given single or multiple (repeated for 10 days) intraperitoneal injections of MAP (5.0 mg/kg/day). The following immune parameters were examined; the number of leukocytes in peripheral blood and the proliferative activity (phytohemagglutinin;PHA, lipopolysaccharide; LPS response) and natural killer (NK) cell activity in splenic lymphocytes. Further, the differences in metabolic function in the spleen in response to MAP (and its metabolite amphetamine) in male and female mice were measured by gas chromatography. The results of the present study were that; 1) single and repeated MAP injections reduced leukocytes; 2) single MAP injection increased the proliferative response of splenic lymphocytes to PHA stimulation in only male mice, but the response to LPS stimulation was slightly increased in both male and female mice; 3) single and repeated MAP injections reduced NK cell activity of splenic lymphocytes, and especially in female mice with 5 injections of MAP; 4) with 10 MAP injections the NK cell activity and leukocytes recovered to the level of controls; and 5) the metabolic activity of MAP was reduced in female mice treated acutely with MAP in comparison to male mice. These results appear to indicate that immune responses to MAP were involved in the different results shown for administration frequency, sex difference and metabolic process of MAP.     

Chemotherapy and the Immune Response in Protozoal Infections1

Available evidence indicates that many of the antiprotozoal drugs currently in use significantly modify the immune response of the host. The effect depends on both the drug and the host. Some drugs enhance the immune response, some are immuno-suppressants, and others enhance some types of immune mechanisms while suppressing others. Future efforts in the development of antiprotozoal drugs should consider their effects on both the parasite and the immune response of the host. Also in the chemotherapy of protozoal infections consideration should be given to the combined usage of immunoenhancing agents and antiprotozoal drugs.     

IL-8 and IDO expression by human gingival fibroblasts via TLRs

Human gingival fibroblasts (HGFs), a predominant cell type in tooth-supporting structure, are presently recognized for their active role in the innate immune response. They produce a variety of inflammatory cytokines in response to microbial components such as LPS from the key periodontal pathogen, Porphyromonas gingivalis. In this study, we demonstrated that HGFs expressed mRNA of TLRs 1, 2, 3, 4, 5, 6, and 9, but not TLRs 7, 8, and 10. Stimulation of HGFs with highly purified TLR2 ligand (P. gingivalis LPS), TLR3 ligand (poly(I:C)), TLR4 ligand (Escherichia coli LPS), and TLR5 ligand (Salmonella typhimurium flagellin) led to expression of IL-8 and IDO. A potent TLR 9 ligand, CpG oligodeoxynucleotide 2006 had no effect, although HGFs showed a detectable TLR9 mRNA expression. No significant enhancement on IL-8 or IDO expression was observed when HGFs were stimulated with various combinations of TLR ligands. Surprisingly, the TLR9 ligand CpG oligodeoxynucleotide 2006 was able to specifically inhibit poly(I:C)-induced IL-8 and IDO expression. TNF-alpha enhanced TLR ligand-induced IL-8 production in HGFs, whereas IFN-gamma enhanced TLR ligand-induced IDO expression. HGF production of IDO in response to P. gingivalis LPS, IFN-gamma, or the two in combination inhibited T cell proliferation in MLRs. The observed T cell inhibition could be reversed by addition of either 1-methyl-dl-tryptophan or l-tryptophan. Our results suggest an important role of HGFs not only in orchestrating the innate immune response, but also in dampening potentially harmful hyperactive inflammation in periodontal tissue.     

Effects of pyrimethamine-sulphadoxine, chloroquine plus chlorpheniramine, and amodiaquine plus pyrimethamine-sulphadoxine on gametocytes during and after treatment of acute, uncomplicated malaria in children

The effects of pyrimethamine-sulphadoxine (PS), chloroquine plus chlorpheniramine, a H1 receptor antagonist that reverses chloroquine resistance in Plasmodium falciparum in vitro and in vivo (CQCP), and amodiaquine plus pyrimethamine-sulphadoxine (AQPS) on gametocyte production were evaluated in 157 children with acute, symptomatic, uncomplicated falciparum malaria who were treated with these drugs. PS was significantly less effective than CQCP or AQPS at clearing asexual parasitaemia or other symptoms of malaria. Gametocyte carriage on days 3, 7, and 14 were significantly higher in those treated with PS. The ratio of the density (per microl blood) of peripheral young gametocyte (PYG), that is, < or = stage III to peripheral mature gametocyte (PMG), that is, stage IV and V, an index of continuing generation of gametocytes, rose to 1 by day 7 of treatment in those treated with PS, but remained consistently below 1 in the other treatment groups. PYG-PMG density ratio increased significantly from day 0-14 in those treated with PS and CQCP (chi2 = 76, P = 0.000001 and chi2 = 42.2, P = 0.00001, respectively) but decreased significantly in those treated with AQPS (chi2 = 53.2, P = 0.000001). Both PS-sensitive and -resistant infections generated PYG (18 of 29 vs 13 of 20, chi2 = 0.04, P = 0.93) but PYG was present only in those with resistant response to CQCP. Combination of PS with amodiaquine (AQ), that is, (AQPS) resulted in less production of PYG, but in this setting, PYG was not indicative of response to AQPS. These data indicate that PS enhanced production or release of young gametocytes when used alone, but generated less young gametocytes when used in combination with AQ. PYG may be used as an indicator of response to CQCP but not PS or PS-based combination drugs.     

Synergistic effect on blastogenesis in murine spleen cells of lipopolysaccharide, lipid A, and acid-degraded polysaccharide from Fusobacterium nucleatum

Interchangeable combinations of Fusobacterium nucleatum Fev1 lipopolysaccharide (LPS) with its split products by acetic acid hydrolysis, i.e. lipid A (LA) and degraded polysaccharide (PS), amplified the blastogenic response in murine spleen cell cultures as measured by [3H]thymidine uptake. Athymic murine spleen cells precultured with LPS-Fev1 for 48 h (stage 1), washed twice and cultured together with fresh cells and either LA or PS for 72 h (stage 2) gave a synergistic response over that found in spleen cell cultures of thymic mice. Spleen cells pre-cultured with LA or PS and with fresh cells and LPS-Fev1 added to stage 2 cultures gave less significant amplification compared with precultures of LPS and either LA or PS together with fresh cells added to stage 2. Precultures with LA, PS or LPS-Fev1 and with pokeweed mitogen (PWM) and fresh cells added produced an additional increment of synergy which was most pronounced in spleen cell cultures of normal mice.     

Functional immune response in Pecten maximus: Combined effects of a pathogen-associated molecular pattern and PAH exposure

Pathogen-associated molecular patterns (PAMPs) enable recognition of structures present in microorganisms such as lipopolysaccharides (LPS). LPS are an essential constituent of the outer membrane of Gram-negative bacteria, stimulating the innate immune system of invertebrates. Here, LPS from Escherichia coli (055:B5) were used to investigate the functional immune response of Pecten maximus after stimulation with a PAMP and to determine the combined effect of a phenanthrene exposure and LPS challenge. Organisms were exposed to 200 μg l^?1 phenanthrene and after 7 d were injected with either physiological saline (injection controls) or LPS solution, and returned to their respective exposure tanks. Haemolymph was sampled from the scallops 48 h post-injection and immune function was assessed using a combination of cellular biological responses. The LPS challenge significantly altered the immune response in P. maximus with increased cell counts and phagocytic activity. An immunosuppressive effect of phenanthrene was also observed in this study; however, exposure to phenanthrene did not significantly impair the organism's ability to respond to a PAMP challenge. The overall level of phagocytosis and cytotoxic capability following the LPS challenge was lower in phenanthrene exposed scallops and may have consequences for disease resistance in this commercially-exploited species.     

Effect of Bordetella pertussis vaccine on the course of lymphocytic choriomeningitis (LCM) virus infection in suckling mice pretreated with dianhydrodulcitol (DAD)

In earlier experiments Bordetella pertussis vaccine was found to enhance and dianhydrodulcitol (DAD) to weaken cellular immune response to intracerebral LCM virus infection in suckling mice. B. pertussis vaccine proved to inhibit the restrictive effect of DAD produced on the immune response in mice when 2-to-4-days-old animals were pretreated with DAD and subsequently, at the age of 16 to 18 days of life, treated with B. pertussis vaccine then infected with LCM virus. Consequently, B. pertussis vaccine enhanced the immune response previously affected by DAD.     

B cell triggering properties of a nontoxic derivative of amphotericin B

The immunomodulating properties of amphotericin B (AMB), an antifungal polyene antibiotic, have been reported in multiple studies. However, many findings on the subject are conflicting, and the precise mechanism of AMB action on the immune system is yet unknown. Because toxicity and limited solubility of AMB are likely to be responsible for these discrepancies, we synthesized a nontoxic derivative of AMB (AMBSH), and we investigated its immune modulating effects on murine B cells. Our results show that AMBSH induces a strong proliferative response under conditions where AMB is weakly efficient or toxic, and that AMBSH supports maturation to Ig secretion. When suboptimal doses of LPS (or BCGF) are present together with AMBSH, a synergistic effect on B cell proliferation occurs. Frequency analyses reveal that, although only a limited number of B cells respond to AMBSH alone, a large population of B cells will respond to subthreshold doses of LPS in the presence of this polyene. Finally, we show that incubation of spleen cells with AMBSH results in an increase in Ia expression. These results are discussed in terms of the membrane disorganizing properties of polyene antibiotics.     

Further development of local IL-2 therapy of cancer: multiple versus single IL-2 treatment of transplanted murine colon carcinoma

We have compared the effect of one and up to four local IL-2 treatments of transplanted MC38 colon carcinoma. A single IL-2 treatment prolonged the survival time (p=0.015), but no cure was obtained. One local IL-2 treatment inhibited tumor growth for about 1 week. After the start of tumor regrowth, a further IL-2 injection was given. After four IL-2 injections 6 out of 13 mice were cured. Histological studies show that IL-2 induced a local vascular leakage syndrome leading to massive peritumoral edema and subsequent necrosis of tumor tissue. IL-2 also attracted infiltrating cells, mainly macrophages. Subsequent IL-2 injections led to complete tumor regression. We believe that the combination of necrotic tumor debris and the IL-2–induced macrophage reaction enhanced a tumor-specific immune response. This local IL-2 application was not toxic.     

HER-2-positive gastric cancer accompanied by liver metastasis: a case report of a rare complete response and literature review

The prognosis of advanced gastric cancer is poor, and the prognosis of late-stage gastric cancer with expression of human epidermal growth factor receptor 2 (HER-2) is worse. Personalized therapy is based on the specific situation of each patient to determine the best treatment plan. We diagnosed a 36-year-old patient with a HER-2-producing adenocarcinoma of the gastric cardia accompanied by liver metastasis. Sixteen cycles of chemotherapy were consecutively administered to the patient, starting with a three-drug combination, followed by a two-drug combination, and finally a single drug as a chemotherapy regimen. During these treatments, the molecular targeted drug trastuzumab was continuously administered. In the end, the patient experienced a complete response (CR). In clinical practice, advanced gastric cancer is not well controlled in certain patients, due to inadequate first-line chemotherapy and its side-effects. If a patient enjoys a strong performance status (PS), we emphasize the importance of molecular-targeted drugs combined with prolonged administration of chemotherapy.