Abundantly Present miRNAs in Milk-Derived Extracellular Vesicles Are Conserved Between Mammals.

Mammalian milk is not only a source of nutrition for the newborn, but also contains various components that regulate further development. For instance, milk is an abundant source of microRNAs (miRNAs), which are evolutionary conserved small non-coding RNAs that are involved in post-transcriptional regulation of target mRNA. MiRNAs present in milk can occur in extracellular vesicles (EVs), which are nanosized membrane vesicles released by many cell types as a means of intercellular communication. The membrane of EVs protects enclosed miRNAs from degradation and harbors molecules that allow specific targeting to recipient cells. Although several studies have investigated the miRNA content in milk EVs from individual species, little is known about the evolutionary conserved nature of EV-associated miRNAs among different species. In this study, we profiled the miRNA content of purified EVs from human and porcine milk. These data were compared to published studies on EVs from human, cow, porcine, and panda milk to assess the overlap in the top 20 most abundant miRNAs. Interestingly, several abundant miRNAs were shared between species (e.g., let-7 family members let-7a, let-7b, let-7f, and miR-148a). Moreover, these miRNAs have been implicated in immune-related functions and regulation of cell growth and signal transduction. The conservation of these miRNA among species, not only in their sequence homology, but also in their incorporation in milk EVs of several species, suggests that they are evolutionarily selected to regulate cell function in the newborn.

The CREATE project: development of certified reference materials for allergenic products and validation of methods for their quantification.

Allergen extracts have been used for diagnosis and treatment of allergy for around 100 years. During the second half of 20th century, the notion increasingly gained foothold that accurate standardization of such extracts is of great importance for improvement of their quality. As a consequence, manufacturers have implemented extensive protocols for standardization and quality control. These protocols have overall IgE-binding potencies as their focus. Unfortunately, each company is using their own in-house reference materials and their own unique units to express potencies. This does not facilitate comparison of different products. During the last decades, most major allergens of relevant allergen sources have been identified and it has been established that effective immunotherapy requires certain minimum quantities of these allergens to be present in the administered maintenance dose. Therefore, the idea developed to introduce major allergens measurements into standardization protocols. Such protocols based on mass units of major allergen, quantify the active ingredients of the treatment and will at the same time allow comparison of competitor products. In 2001, an EU funded project, the CREATE project, was started to support introduction of major allergen based standardization. The aim of the project was to evaluate the use of recombinant allergens as reference materials and of ELISA assays for major allergen measurements. This paper gives an overview of the achievements of the CREATE project.

Long-term treatment with allergoid immunotherapy with Parietaria. Clinical and immunologic effects in a randomized, controlled trial.

BACKGROUND: Specific immunotherapy (SIT) is a valuable treatment for respiratory allergy, and the use of chemically modified allergens (allergoids) has improved its safety, as testified by several studies. We evaluated the effects of a SIT course with an allergoid extract of Parietaria pollen in a double-blind, placebo-controlled trial. METHODS: The study was double-blind in the first year; then it was prolonged up to 3 years with all patients on active treatment. Clinical effectiveness, safety, skin reactivity, systemic immunologic parameters, and subjective assessment were evaluated. We also had available a self-evaluation recorded in a follow-up visit 4 years after the discontinuation of SIT. RESULTS: A significant reduction of the symptoms plus drug intake scores during the pollen seasons was observed in the patients receiving active SIT. The placebo patients, after switching to active SIT, also showed significant clinical improvement. The clinical efficacy persisted during years 2 and 3 of treatment. After year 1, the actively treated patients reported a significant subjective improvement (frequency of symptoms, P = 0.001; duration of symptoms, P = 0.024; physical performance, P = 0.043) compared with the placebo group. The self-evaluation by visual analog scale showed that all patients maintained a significant clinical improvement up to 4 years after discontinuing SIT (year 1: active=+31.6%, placebo=-15.7%; year 7: active=+35.8%, placebo=+31.3%). The systemic immunologic changes after active SIT paralleled those described elsewhere (IgE decreased from 22 to 9 and from 21 to 8 IU/ml; IgG4 increased from 43 to 87 and from 18 to 60 IU/ml). A significant decrease in skin reactivity to three different allergen concentrations was observed at year 3 compared with pretreatment values (P<0.05). CONCLUSIONS: The investigational SIT with Parietaria appeared to be effective and safe; a 3-year course of treatment achieved a long-lasting efficacy.

Measurement of IgE antibodies against purified grass pollen allergens (Lol p 1, 2, 3 and 5) during immunotherapy.

BACKGROUND: IgE titres tend to rise early after the start of immunotherapy, followed by a decline to pre-immunotherapy levels or lower. OBJECTIVES: We were interested to know whether the early increase in IgE antibodies includes new specificities of IgE, and whether these responses persist. METHODS: Sera of 64 patients undergoing grass pollen immunotherapy were tested for IgE against four purified grass pollen allergens: Lol p 1, 2, 3, and 5. At least two serum samples were taken, one before the start of therapy and one between 5 and 18 months after the first immunization (mean: 10 months). RESULTS: The mean IgE responses to Lol p 1, 2 and 3 showed a moderate but not significant increase. In contrast, the mean IgE response to Lol p 5 showed a significant decrease of > 30%. IgE against total Lohum perenne pollen extract moderately increased (> 20%), showing that a RAST for total pollen is not always indicative for the development of IgE against its major allergens. For > 40% of the patients it was found that IgE against one or more of the four allergens increased, while IgE against the remaining allergen(s) decreased. For 10 sera the ratio of IgE titres against at least two allergens changed by at least a factor of 5. The changes in specific IgE also included conversions from negative (< 0.1 RU) to positive (0.6 to 5.0 RU) for five patients. For two patients, the induction of these 'new' IgE antibodies against major allergens was shown to result in a response that was persistent over several years. CONCLUSION: Although active induction of new IgE specificities by immunotherapy was not really proven, the observations in this study indicate that monitoring of IgE against purified (major) allergens is necessary to evaluate changes in specific IgE in a reliable way.

Grass pollen immunotherapy induces highly cross-reactive IgG antibodies to group V allergen from different grass species.

Sera from two groups of patients receiving grass pollen immunotherapy were tested on IgG reactivity with group V allergen from six different grass species. One group of patients was treated with a mixture of 10 grass species, and the other with a mixture of five. Only Lolium perenne, Dactylis glomerata, and Phleum pratense were present in both mixtures. Although Anthoxanthum odoratum and Secale cereale were absent from the mixture of five, IgG responses to Ant o V and Sec c V were comparable in both patient groups. This reactivity was inhibited for 92-99% with L. perenne extract, illustrating the cross-reactive nature of the IgG antibodies. The presence of A. odoratum and S. cereale in the mixture resulted in only minor amounts of species-specific anti-group V IgG. These results indicate that application of just one grass species in immunotherapy might be sufficient to induce an IgG response that covers other relevant Gramineae species as well.

[Therapy of hayfever and pollen asthma in children with a new, modified allergen extract]. / Therapie von Heuschnupfen und Pollenasthma bei Kindern mit einem neuen, modifizierten Allergenextrakt.

During a period of 3 years a controlled and prospective study was performed, in which 20 children with pollinosis and pollen asthma (grass pollen) were treated either with a new modified allergenic extract (preparation I: Purethal) or with a standard semi-depot extract (preparation II: Depot-HAL) from the same producer. During the pollen season symptoms, side effects, additional medication, and pollen counts were registered. 20 patients were treated perennially: 5 with preparation II and with reduction of the doses during the pollen season (group A: treatment during 3 successive years), 15 without reduction of the doses during te season with preparation I (group B, 8 patients: treatment during 3 successive years; group C, 7 patients: treatment during 2 successive years). Patients in all groups showed more symptoms in May and June. In the groups B and C less symptoms were recorded. Especially, the score of asthma symptoms decreased. However, the need for additional medication was somewhat higher in these groups as compared to group A. The differences were not statistically significant. In 50% of the children no local side reactions were observed after the subcutaneous injections. In the groups B and C the number of late local reactions after 6 to 8 hours was somewhat higher. The patients treated with preparation I did not show an increase of allergen-specific IgG during the pollen season. The relative contribution of allergen-specific IgG1 and IgG4 to total specific IgG was lower in the blood samples of these patients as compared to those of group A. The efficacy and the therapeutic safety of the two preparations are comparable. Preparation I has a number of practical advantages, especially in the treatment of children.

Changes in nuclear protein pattern by glucocorticoid treatment of lymphoid cells.

Glucocorticoids initiate a cytolytic process in lymphoid cells. The ultimate response is preceded by several phenomena. It is generally accepted that these are mediated through messenger proteins. The induction of these proteins is considered the primary effect of glucocorticoids. However, as yet specific gene products have not been identified. In electrophoretic assays, we observed an increased concentration of 6 nuclear proteins within a few hours of exposure of lymphoid cells to glucocorticoids. These proteins displayed prominent DNase activity. However, further studies showed: (1) that the proteins concerned are histones, (2) that histones are more easily extracted after glucocorticoid-induced alterations of lymphoid cells, and (3) that basic proteins in general express nuclease activity under certain experimental conditions. This nuclease activity is, however, artifactual. Therefore, though the changes observed are certainly related to glucocorticoid-induced effects, these do not reflect the induction of specific proteins. The results of the study indicate that glucocorticoid-induced changes in the concentration of cellular proteins should be interpreted with caution.

Mitochondrial biogenesis during the activation of lymphocytes by mitogens: the immunosuppressive action of tetracyclines.

The role of mitochondrial biogenesis and function during mitogenic stimulation of rat thymocytes was investigated. The results show that mitochondrial biogenesis is required to provide the ATP for the energy-requiring processes occurring during blastogenesis. Impairment of mitochondrial biogenesis by inhibition of mitochondrial protein synthesis inhibits blast transformation. Since the tetracyclines impair mitochondrial protein synthesis, the results offer an explanation for the well-known immunosuppressive effects of these antibiotics.

Inhibition of mitochondrial protein synthesis influences the glucocorticoid sensitivity of lymphoid cells.

Inhibition of mitochondrial protein synthesis impairs the formation of the 13 polypeptides encoded on the mitochondrial genome. These polypeptides are part of enzyme complexes involved in oxidative phosphorylation. Prolonged inhibition of mitochondrial protein synthesis thus reduces the oxidative phosphorylation capacity which ultimately results in impairment of energy-requiring processes. Via a different mechanism glucocorticoid hormones also decrease the oxidative phosphorylation capacity of, e.g., lymphoid cells. The present study shows that inhibition of mitochondrial protein synthesis influences glucocorticoid-induced responses of lymphoid cells in two opposing manners. (a) It is enhanced after induction in cells with a reduced oxidative phosphorylation capacity resulting from preceding inhibition of mitochondrial protein synthesis. This can be explained by the synergistic effects of glucocorticoids and prolonged inhibition of mitochondrial protein synthesis on energy-producing processes. (b) It is counteracted when mitochondrial protein synthesis is impaired during induction of the response. The latter observation suggests that mitochondrial protein synthesis is involved in the generation of glucocorticoid-induced effects on lymphoid cells.

Doxycycline in combination chemotherapy of a rat leukemia.

Inhibition of mitochondrial protein synthesis by doxycycline (DC), a tetracycline analogue, has significant antitumor effects in several tumor systems. In the present study, the effects of continuous DC treatment combined with intermittent administration of Adriamycin or 1-beta-D-arabinofuranosyl cytosine on the growth of a rat leukemia were investigated. The presence of DC retards tumor relapse after 1-beta-D-arabinofuranosyl cytosine or Adriamycin treatment significantly. DC may therefore be of value in several modalities of antitumor treatment.

Mitochondrial biogenesis and mitochondrial activity during the progression of the cell cycle of human leukemic cells.

Mitochondrial (mt) biogenesis and mt function were investigated during the cell cycle of leukemic cells. The study shows that the activity of enzymes involved in oxidative phosphorylation increases in the early G1 phase. This increase in activity precedes that of other mt enzymes such as citrate synthase and adenylate kinase. Therefore, the synthesis of mt enzymes, needed for the reduplication of the mt mass in the course of the cell cycle, occurs in a sequential order. The enzymes of the system for oxidative phosphorylation are composed of several subunits. Some of these subunits are encoded on mtDNA and synthesized by mt-specific RNA and protein synthesis. This explains why inhibition of mt protein synthesis during the progression of the cell cycle of G1-enriched cells results in an increasing shortage of ATP. This lack of ATP results first in progression delay and, subsequently, in a cell cycle block in early G1. Furthermore, shortage of ATP impairs the increase in activity of at least one mt matrix enzyme. This study offers new information about a number of aspects of mt biogenesis and mt function during cell cycle progression and elucidates the cytostatic mechanism resulting from prolonged inhibition of mt protein synthesis.

Different effects of oxytetracycline and doxycycline on mitochondrial protein synthesis in rat liver after long-term treatment.

The tetracyclines inhibit specifically mitochondrial (mt) and bacterial protein synthesis when they are present in low concentrations (2-10 micrograms/ml). There is no difference between the various members of this group of antibiotics in this respect. In the present study, however, it is shown that the inhibitory effect of doxycycline on mt-protein synthesis in rat liver is partially lost after continuous treatment for more than 1 week, whereas oxytetracycline continues to inhibit mt-protein synthesis effectively after 1 week of treatment. To find an explanation for this difference between doxycycline and oxytetracycline, a detailed study was made of the distribution and the effects on mt-protein synthesis of both tetracyclines under various conditions in rat liver. The results of the studies lead to the hypothesis that doxycycline treatment induces the formation of a doxycycline complex, and thus to a reduced amount of free doxycycline. This may explain the loss of effective inhibition of mt-protein synthesis.

The effect of inhibition of mitochondrial protein synthesis on the proliferation and phenotypic properties of a rat leukemia in different stages of in-vivo tumor development.

It has been shown before that prolonged treatment with doxycycline (DC), an inhibitor of mitochondrial protein synthesis, leads to proliferation arrest of a leukemia in the rat and, moreover, to eradication of this tumor. It has also been demonstrated that the period of treatment required to achieve this is shorter when DC administration is started in later stages of tumor progression. Therefore, the leukemic cells may have properties with regard to DC sensitivity which change with time during tumor progression. In the present study this hypothesis was tested by studying the permeability for DC, the presence of cell-surface molecules, and the mitochondrial content of the leukemic cells in various stages of tumor development in control and in DC-treated rats. Changes in DC permeability or antigenic phenotype were not observed, but the content of mitochondria decreases during tumor progression. DC treatment leads to an additional reduction of the content of functional mitochondria which results in proliferation arrest. The higher mitochondrial content of the leukemic cells during the earlier stages of tumor development explains thus why a longer period of DC treatment is needed to achieve growth arrest when treatment is started in these stages.

Biogenesis of mitochondria and genetics of mitochondrial defects.

Mitochondria are formed by the concerted action of two genetic systems: the nucleocytoplasmic system and the intrinsic mitochondrial system. The genetic contribution of the mitochondria is modest because the genetic potential of mtDNA of mammals is restricted to the equivalent of about 16,000 base pairs. For various animals and man the complete base sequence of mtDNA is known and all possible polypeptide genes have now been assigned to subunits of the respiratory enzymes. The mtDNA sequences are not present on the nuclear genome. From a genetic point of view it is important that the inheritance of mtDNA is strictly maternal. Mutations of mtDNA primarily lead to impairments of energy metabolism. In view of the indispensability of oxidative phosphorylation for obligatory aerobic organisms, such mutations should be lethal. However, there are various inborn errors of metabolism with tissue-specific manifestations, which are maternally inherited. The question discussed is whether these diseases can be explained on the basis of mutations of mitochondrial gene products. Tissue specificity poses a special problem, since it is not very attractive to assume that there is a heterogenous population of mtDNA molecules in the fertilized egg. Therefore, one should rather think in terms of a double mutational event, one tissue-specific cytoplasmic and the other general mitochondrial. These mutations only give rise to metabolic disturbances if they are expressed together in the same cell.

Inhibition of mitochondrial protein synthesis leads to proliferation arrest in the G1-phase of the cell cycle.

Mitochondrial protein synthesis is specifically inhibited by low concentrations of tetracyclines. Prolonged inhibition of mitochondrial protein synthesis leads to a lack of oxidative ATP generating capacity, which results in proliferation arrest of normal and malignant cells of epithelial origin, as has been shown previously. The present study indicates that this holds true also for fibroblasts and sarcoma cells. It is shown that this proliferation arrest leads to accumulation of the growth-arrested cells in the G1-phase of the cell cycle. This offers several interesting possibilities to use tetracyclines in anticancer combination therapies.

Arrest of the proliferation of renal and prostate carcinomas of human origin by inhibition of mitochondrial protein synthesis.

The results described in this paper demonstrate that proliferation arrest by low concentrations of tetracyclines, which has previously been shown in experiments with animal tumor systems, can also be achieved in tumor systems of human origin. Tetracyclines specifically inhibit mitochondrial protein synthesis. Prolonged and continuous impairment of protein synthesis inside the mitochondria leads to reduction of the cellular concentration of the polypeptide products which are coded and synthesized within mitochondria. These products are part of the oxidative phosphorylative system of the cell. Long-term tetracycline treatment leads to a decrease of oxidative ATP-generating capacity as monitored by cytochrome c oxidase activity. This may cause severe energetic or metabolic disturbances which explain the proliferation arrest observed. Proliferation arrest, provided that mitochondrial protein synthesis is blocked effectively, is found in vitro as well as in vivo. It is shown that the effect of doxycycline is not limited to cytostasis; prolonged doxycycline treatment is clearly cytotoxic for the tumor cells.

The effect of doxycycline on polyvinylpyrrolidone-induced granuloma formation in the rat liver.

The tetracyclines specifically inhibit mitochondrial protein synthesis when present at the same low concentrations as used for their antibacterial action. Inhibition of mitochondrial protein synthesis leads to decrease in the oxidative energy-generating capacity of cells. Therefore, the presence of tetracyclines may result in proliferation arrest. In the present study we show that continuous intravenous administration of polyvinylpyrrolidone (PVP) induces the formation of granulomas in the normal rat liver; the rats usually die within 2 weeks of continuous PVP treatment. Athymic (nude) rats appear to be more resistent to the deleterious effects of PVP as they survive the treatment for at least 5 weeks. Although the livers of the PVP-treated nude rats are heavily infiltrated with phagocytic cells, they seldom show granulomas. Reconstitution of nude rats with syngenic thymocytes leads, on the other hand, to extensive granuloma formation. Normal rats treated continuously with PVP plus doxycycline, however, all survive, their livers showing only a few very small granulomas and the normal low number of phagocytic cells. We conclude that the formation of granulomas induced by PVP is a process which is mediated by T-lymphocytes. Because doxycycline prevents this kind of granuloma formation it seems likely that doxycycline not only impairs the proliferation and differentiation of T-lymphocytes but also of monocytes and macrophages.

Specific inhibition of mitochondrial protein synthesis influences the amount of complex I in mitochondria of rat liver and Neurospora crassa directly.

Specific inhibition of mitochondrial protein synthesis reduces the oxidation rate of NADH-linked substrates in rat liver as well as in Neurospora crassa mitochondria. The present study shows that this is due to the fact that inhibition of mitochondrial protein synthesis leads to a decrease of the concentration of active complex I. Therefore, these results demonstrate that at least one of the genes for the subunits of complex I is localized on mitochondrial DNA.