Oral administration of Debaryomyces hansenii CBS8339-ß-glucan induces trained immunity in newborn goats.

Beta-glucans from yeast can induce trained immunity in in vitro and in vivo models. Intraperitoneal doses of ß-glucans in mammals have shown to induce trained immunity, but the training effects of orally administering ß-glucans are unknown. Newborn goats are susceptible to infections in the neonatal stage, so the induction of trained immunity could improve animal survival. This study aimed to describe the in vitro effects of immunological training by ß-glucan from Debaryomyces hansenii (ß-Dh) on caprine monocytes, as well as its in vivo effects using oral doses on newborn goats upon challenge with lipopolysaccharide (LPS). Hence in vitro, goat monocytes trained with ß-Dh up-regulated the gene expression of macrophage surface markers (CD11b and F4/80) whereas enhanced cell survival and high phagocytic ability was found upon LPS challenge. In the in vivo experiment, newborn goats stimulated with two doses (day -7 and - 4) of ß-Dh (50 mg/kg) and challenged (day 0) with LPS showed an increase in respiratory burst activity, IL-1ß, IL-6, and TNFα production in plasma, and transcription of the macrophage surface markers. This study has demonstrated for the first time that trained immunity was induced with oral doses of ß-glucan upon LPS challenge in mammals using newborn goats.

Constitutive TNF-α signaling in neonates is essential for the development of tissue-resident leukocyte profiles at barrier sites.

Newborn infants have a high disposition to develop systemic inflammatory response syndromes (SIRSs) upon inflammatory or infectious challenges. Moreover, there is a considerable trafficking of hematopoietic cells to tissues already under noninflammatory conditions. These age-specific characteristics suggest a hitherto unappreciated crucial role of the vascular endothelium during the neonatal period. Here, we demonstrate that healthy neonates showed already strong endothelial baseline activation, which was mediated by a constitutively increased production of TNF-α. In mice, pharmacological inhibition of TNF-α directly after birth prevented subsequent fatal SIRS but completely abrogated the recruitment of leukocytes to sites of infection. Importantly, in healthy neonates, blocking TNF-α at birth disrupted the physiologic leukocyte trafficking, which resulted in persistently altered leukocyte profiles at barrier sites. Collectively, these data suggest that constitutive TNF-α-mediated sterile endothelial activation in newborn infants contributes to the increased risk of developing SIRS but is needed to ensure the postnatal recruitment of leukocytes to organs and interfaces.-Bickes, M. S., Pirr, S., Heinemann, A. S., Fehlhaber, B., Halle, S., Völlger, L., Willers, M., Richter, M., Böhne, C., Albrecht, M., Langer, M., Pfeifer, S., Jonigk, D., Vieten, G., Ure, B., von Kaisenberg, C., Förster, R., von Köckritz-Blickwede, M., Hansen, G., Viemann, D. Constitutive TNF-α signaling in neonates is essential for the development of tissue-resident leukocyte profiles at barrier sites.

Probiotic effects of marine Debaryomyces hansenii CBS 8339 on innate immune and antioxidant parameters in newborn goats.

Several marine Debaryomyces hansenii strains have shown probiotic effects on aquatic animals, and D. hansenii-derived ß-glucans have recently provided immunostimulant effects on goat leukocytes. This study assessed the probiotic effects of live yeast D. hansenii CBS 8339 on newborn goats administered orally, and subsequently challenged in vitro with Escherichia coli. D. hansenii CBS 8339 demonstrated the capacity to survive gastrointestinal tract conditions (bile salts and acid pH tolerance) and adhere to goat intestine. Twelve Saanen × Nubian crossbred newborn goats (2.9 ± 0.47 kg) were fed with a controlled diet or D. hansenii (0.7 g/kg body weight per day)-supplemented milk for 30 days. Blood samples of newborn goats were taken at days 15 and 30, and peripheral blood leukocytes were isolated for bacterial challenge, and immunological and antioxidant analyses. Despite cell viability was higher in leukocytes of goat kids fed with the yeast supplement, protection against E. coli challenge was not significantly affected. On the other hand, at day 15, oral administration of D. hansenii enhanced respiratory burst and catalase activity and increased superoxide dismutase activity after challenge. In contrast, at day 30, administration of the yeast supplement increased peroxidase activity and enhanced nitric oxide production and catalase activity after challenge. Finally, the yeast-supplemented diet upregulated the expression of the receptor genes TLR (2, 4, 6), modulator genes Raf.1, Syk, and Myd88, transcription factor gene AP-1, and cytokine genes IL-1ß and TNF-α only at day 15 in leukocytes from unchallenged goat kids. These results demonstrated that a short time (15 days) of orally administering the probiotic D. hansenii CBS 8339 to newborn goats stimulated innate immune and antioxidant parameters and the expression of immune-related gene signaling pathways.

Testicular cells exhibit similar molecular responses to cigarette smoke condensate ex vivo and in vivo.

Male exposure to cigarette smoke is associated with seminal defects and with congenital anomalies and childhood cancers in offspring. In mice, paternal exposure to cigarette smoke condensate (CSC) causes molecular defects in germ cells and phenotypic effects in their offspring. Here we used an ex vivo testicular explant model and in vivo exposure to determine the concentration at which CSC impairs spermatogenesis and offspring development. We explanted testis tissue at postnatal day (P)5.5 and cultured it until P11.5. Assessment of growth parameters by analyzing expression of cell-specific markers revealed that the explant system maintained structural and functional integrity. We exposed the P5.5 to -11.5 explants to various concentrations (40-160 µg/ml) of CSC and confirmed that nicotine in the CSC was metabolized to cotinine. We assessed various growth and differentiation parameters, as well as testosterone production, and observed that many spermatogenesis features were impaired at 160 µg/ml CSC. The same parameters were impaired by a similar CSC concentration in vivo Finally, females mated to males that were exposed to 160 µg/ml CSC neonatally had increased rates of pup resorption. We conclude that male exposure to CSC impairs offspring development and that the concentration at which CSC impairs spermatogenesis is similar in vivo and ex vivo. Given that the concentrations of CSC we used contained similar doses of nicotine as human smokers are exposed to, we argue that our model mimics human male reproductive effects of smoking.-Esakky, P., Hansen, D. A., Drury, A. M., Felder, P., Cusumano, A., Moley, K. H. Testicular cells exhibit similar molecular responses to cigarette smoke condensate ex vivo and in vivo.

Postnatal growth after intrauterine growth restriction alters central leptin signal and energy homeostasis.

Intrauterine growth restriction (IUGR) is closely linked with metabolic diseases, appetite disorders and obesity at adulthood. Leptin, a major adipokine secreted by adipose tissue, circulates in direct proportion to body fat stores, enters the brain and regulates food intake and energy expenditure. Deficient leptin neuronal signalling favours weight gain by affecting central homeostatic circuitry. The aim of this study was to determine if leptin resistance was programmed by perinatal nutritional environment and to decipher potential cellular mechanisms underneath.We clearly demonstrated that 5 months old IUGR rats develop a decrease of leptin sentivity, characterized by no significant reduction of food intake following an intraperitoneal injection of leptin. Apart from the resistance to leptin injection, results obtained from IUGR rats submitted to rapid catch-up growth differed from those of IUGR rats with no catch-up since we observed, for the first group only, fat accumulation, increased appetite for food rich in fat and increased leptin synthesis. Centrally, the leptin resistant state of both groups was associated with a complex and not always similar changes in leptin receptor signalling steps. Leptin resistance in IUGR rats submitted to rapid catch-up was associated with alteration in AKT and mTOR pathways. Alternatively, in IUGR rats with no catch-up, leptin resistance was associated with low hypothalamic expression of LepRa and LepRb. This study reveals leptin resistance as an early marker of metabolic disorders that appears before any evidence of body weight increase in IUGR rats but whose mechanisms could depend of nutritional environment of the perinatal period.

The role of neuronal connexins 36 and 45 in shaping spontaneous firing patterns in the developing retina.

Gap junction coupling synchronizes activity among neurons in adult neural circuits, but its role in coordinating activity during development is less known. The developing retina exhibits retinal waves--spontaneous depolarizations that propagate among retinal interneurons and drive retinal ganglion cells (RGCs) to fire correlated bursts of action potentials. During development, two connexin isoforms, connexin 36 (Cx36) and Cx45, are expressed in bipolar cells and RGCs, and therefore provide a potential substrate for coordinating network activity. To determine whether gap junctions contribute to retinal waves, we compared spontaneous activity patterns using calcium imaging, whole-cell recording, and multielectrode array recording in control, single-knock-out (ko) mice lacking Cx45 and double-knock-out (dko) mice lacking both isoforms. Wave frequency, propagation speed, and bias in propagation direction were similar in control, Cx36ko, Cx45ko, and Cx36/45dko retinas. However, the spontaneous firing rate of individual retinal ganglion cells was elevated in Cx45ko retinas, similar to Cx36ko retinas (Hansen et al., 2005; Torborg and Feller, 2005), a phenotype that was more pronounced in Cx36/45dko retinas. As a result, spatial correlations, as assayed by nearest-neighbor correlation and functional connectivity maps, were significantly altered. In addition, Cx36/45dko mice had reduced eye-specific segregation of retinogeniculate afferents. Together, these findings suggest that although Cx36 and Cx45 do not play a role in gross spatial and temporal propagation properties of retinal waves, they strongly modulate the firing pattern of individual RGCs, ensuring strongly correlated firing between nearby RGCs and normal patterning of retinogeniculate projections.

Neonatal BCG immunization followed by DNAhsp65 boosters: highly immunogenic but not protective against tuberculosis - a paradoxical effect of the vector?

A new tuberculosis vaccine is urgently needed. Prime-boost strategies are considered very promising and the inclusion of BCG is highly desirable. In this investigation, we tested the protective efficacy of BCG delivered in the neonatal period followed by boosters in the adult phase with a DNA vaccine containing the hsp65 gene from Mycobacterium leprae (pVAXhsp65). Immune responses were characterized by serum anti-hsp65 antibody levels and IFN-gamma and IL-5 production by the spleen. Amounts of these cytokines were also determined in lung homogenates. Protective efficacy was established by the number of colony-forming units (CFU) and histopathological analysis of the lungs after challenge with Mycobacterium tuberculosis. Immunization with BCG alone triggered a significant reduction of CFU in the lungs and also clearly preserved the pulmonary parenchyma. BCG priming also increased the immunogenicity of pVAXhsp65. However, boosters with pVAXhsp65 or the empty vector abolished the protective efficacy of BCG. Also, higher IL-5 levels were produced by spleen and lungs after DNA boosters. These results demonstrated that neonatal BCG immunization followed by DNAhsp65 boosters is highly immunogenic but is not protective against tuberculosis.

Developmental changes in the fibre population of the optic nerve follow an avian/mammalian-like pattern in the turtle Mauremys leprosa.

The changes in the axon and growth cone numbers in the optic nerve of the freshwater turtle Mauremys leprosa were studied by electron microscopy from the embryonic day 14 (E14) to E80, when the animals normally hatch, and from the first postnatal day (P0) to adulthood (5 years on). At E16, the first axons appeared in the optic nerve and were added slowly until E21. From E21, the fibre number increased rapidly, peaking at E34 (570,000 fibres). Thereafter, the axon number decreased sharply, and from E47 declined steadily until reaching the mature number (about 330,000). These observations indicated that during development of the retina there was an overproduction and later elimination of retinal ganglion cells. Growth cones were first observed in the optic nerve at as early as E16. Their number increased rapidly until E21 and continued to be high through E23 and E26. After E26, the number declined steeply and by E40 the optic nerve was devoid of growth cones. These results indicated that differentiation of the retinal ganglion cells occurred during the first half of the embryonic life. To examine the correlation between the loss of the fibres from the optic nerve and loss of the parent retinal ganglion cells, retinal sections were processed with the TUNEL technique. Apoptotic nuclei were detected in the ganglion cell layer throughout the period of loss of the optic fibres. Our results showed that the time course of the numbers of the fibres in the developing turtle optic nerve was similar to those found in birds and mammals.

Effect of Bacillus licheniformis and Bacillus subtilis supplementation of ewe's feed on sheep milk production and young lamb mortality.

The purpose of this pilot study was to evaluate under field conditions the effect of a probiotic containing Bacillus licheniformis and Bacillus subtilis on young lamb mortality and sheep milk production when administered in the late pregnancy and lactation feed of ewes. In a sheep farm, two groups of milking ewes with identical genetic material, management, nutrition, health status and similar production characteristics were formed. One group (46 ewes) served as control, while the other one (48 ewes) served as a probiotic-treated group. Both groups of ewes received a similar feeding regiment, but the ewes of the second group were additionally offered a probiotic product containing B. licheniformis and B. subtilis (BioPlus 2B, Chr. Hansen, Denmark) at the approximate dose of 2.56 x 10(9) viable spores per ewe per day. Lamb mortality during the 1.5 months suckling period, and milk yield during the 2 months of milk collection for commercial purposes have been recorded. In the non-treated control group, 13.1% mortality was observed versus 7.8% in the probiotic-treated group (P = 0.33), with mortality being mainly due to diarrhoea. Microbiological examination of diarrhoeic faeces from some of the dead lambs in both groups revealed the presence of Escherichia coli. The average daily milk yield per ewe was significantly lower in the control group (0.80 l) than that in the probiotic-treated group (0.93 l) (P < 0.05). Fat and protein content of milk in ewes that received probiotics was significantly (P < 0.05) increased compared with untreated ewes. It was concluded that supplementing ewe's feed with probiotics may have beneficial effect on subsequent milk yields, fat and protein content.

Heat shock protein Hsp60-reactive gamma delta cells: a large, diversified T-lymphocyte subset with highly focused specificity.

Previously, we detected a subset of gamma delta T cells in the newborn mouse thymus that responded to the mycobacterial heat shock protein Hsp60, as well as with what seemed to be a self-antigen. All of these cells expressed V gamma 1, most often in association with V delta 6+. It was not clear, however, whether similar, mature gamma delta cells with Hsp60 reactivity are common outside of the thymus, or rather, whether they are largely eliminated during development. From the data presented here, we estimate that gamma delta cells responding to Hsp60 comprise 10-20% of normal splenic and lymph node gamma delta T cells. Such cells, derived from adult spleen, always express a V gamma 1-J gamma 4-C gamma 4 gamma chain, although not all cells with this gamma chain show Hsp60 reactivity. Many of these V gamma 1+ cells also express V delta 6-J delta 1-C delta, though fewer than in V gamma 1+ cells from the newborn thymus. Extensive diversity is evident in both the gamma and delta chain junctional amino acids of the receptors of these cells, indicating that they may largely develop in the thymus of older animals or undergo peripheral expansion. Finally, we found that all such cells responding to both a putative self-antigen and to mycobacterial Hsp60 respond to a 17-amino acid synthetic peptide representing amino acids 180-196 of the Mycobacterium leprae Hsp60 sequence. This report demonstrates that a large subset of Hsp60-reactive peripheral lymphoid gamma delta T cells preexists in normal adult mice, all members of which respond to a single segment of this common heat shock protein.

In vitro interaction of M. leprae-infected Schwann cells and splenic cells.

The interaction between M. leprae-infected cultured Schwann cells and sensitized splenic cells was noted both under light and electron microscopy. No evidence of cytomorphological changes in infected Schwann cells was obtained. However, sensitized splenic cells were noted to undergo degenerative changes suggestive of the phenomenon of apoptosis. Subsequently a large number of these degenerated cells were observed within the Schwann cell. Such a process has not been hitherto reported in the histopathology of leprous nerves. Nevertheless, these findings indicate an aberrant metabolic function in M. leprae-infected Schwann cells.

Gamma interferon, but not Mycobacterium leprae, induces major histocompatibility class II antigens on cultured rat Schwann cells.

In order to investigate the possible role of Schwann cells in immune reactions, and in particular their involvement in the response to infection with Mycobacterium leprae, it was determined under what conditions Schwann cells express major histocompatibility complex class II (MHC class II) antigens, since these molecules are thought to have a key role in antigen presentation during cellular immune responses. In situ and in vitro preparations from newborn and adult rat sciatic nerves were used as a model system to examine this question. Schwann cells in dissociated cell cultures did not express immunohistochemically detectable amounts of MHC class II antigens. Teased nerve preparations from the sciatic nerves of healthy adult rats showed no detectable immunolabelling of either myelin-forming or non-myelin-forming Schwann cells. When dissociated Schwann cell cultures derived from the sciatic nerves of either neonatal or adult rats were treated with 10, 50 or 100 units of gamma interferon, MHC class II antigens were detectable on the surface of some Schwann cells 48 h after addition of the interferon. By 72 h, 32.29 +/- 3.9% of Schwann cells in the cultures from neonatal rats and 53.32 +/- 5.4% of Schwann cells in cultures from adult rats, identified by the presence of intracellular S-100, were clearly MHC class II-positive, especially at doses of 50 and 100 units per ml of gamma interferon. Some, but not all, of the fibroblastic cells were very weakly MHC class II-positive. Infection of the cultures with Mycobacterium leprae did not induce MHC class II antigen expression in either Schwann cells or fibroblasts.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationship between T-cell population in neonatally thymectomized Lewis rats and susceptibility to infection with mycobacterium leprae.

The neonatally thymectomized Lewis rat (NTLR) is highly susceptible to infection with M. leprae. However, a significant percentage of NTLR respond to infection with M. leprae in much the same way as do intact rats, yet show no evidence of residual thymus. To determine whether there was a correlation between the number of remaining T-cells and susceptibility to infection with M. leprae, a direct fluorescent antibody test was performed using a highly specific, absorbed antithymocyte globulin labeled with fluorescein isothiocyanate. Both total circulating white blood cells and T-cells were significantly depressed in all NTLR examined. Although the greatest numbers of M. leprae were found in NTLR from the groups having the lowest percentage of circulating T-cells, these groups also contained NTLR infected with small numbers of M. leprae. The groups containing NTLR with the highest percentages of circulating T-cells also contained animals with both moderate and severe M. leprae infection. The response of cultured splenic lymphocytes from NTLR and normal rats to the T-cell mitogen concanavalin A was investigated to determine whether there was any correlation between T-cell activity and susceptibility to M. leprae infection. The mean stimulation index for normal rats was five to ten times greater than indices for NTLR, but there were no significant differences between NTLR with a well developed, generalized infection and those with a poorly developed infection. it was concluded that since there was no apparent relationship between T-cell depletion and susceptibility to infection with M. leprae, an additional, unknown mechanism was also involved.

Neonatally thymectomized Lewis rats infected with Mycobacterium leprae. 2. Histopathologic and electron microscopic observations.

We report the histologic and electron microscopic findings following intravenous inoculation of M. leprae into neonatally thymectomized Lewis rats, which were killed one to two years later. All organs appeared normal grossly. Histologic changes were confined to the footpads, snout, ears, tail, and testes, all of which were involved in every rat. The tissues were edematous and infiltrated by varying numbers of foamy macrophages. In the footpads muscle fibers were vacuolated, and small nerves showed degenerative changes. Large numbers of M. leprae were present in macrophages and striated muscle cells and smaller numbers in perineural cells and pericytes, as well as lying free in the tissues. Occasional intracellular bacilli were found throughout the reticuloendothelial system. Electron microscopy confirmed that the majority of organisms were within activated macrophages. Both intact and fragmented bacilli were contained within double-membrane bound vacuoles. Numerous M. leprae were lying free within the sarcoplasm of striated muscle cells. Virtually all of the extracellular organisms were degenerating.

Dapsone chemotherapy of Mycobacterium leprae infection of the neonatally thymectomized Lewis rat.

In order to learn whether the neonatally thymectomized Lewis rat (NTLR) infected with Mycobacterium leprae could serve as a model for chemotherapeutic studies in a situation resembling that found in human lepromatous leprosy, NTLR inoculated with M. leprae either locally or intravenously 9 to 16 months earlier were treated for from 1.5 to 8.5 months with dapsone (4,4'-diaminodiphenylsulfone, DDS) incorporated in the rat chow in the concentration providing the minimal inhibitory concentration of the drug for M. leprae and in the 100-fold larger concentration. NTLR were killed at intervals; the M. leprae were counted and passed to mice. Treatment with the smaller dosage of dapsone neither killed M. leprae nor reduced the number of organisms in the bacterial populations, whereas treatment with the larger dosage both killed M. leprae and reduced their numbers. The rate at which the organisms were killed (i.e., rendered noninfective for mice) was much the same as that in patients treated with dapsone in comparable dosage. The dead organisms were removed from the rat tissues at a faster rate than encountered in patients. The NTLR may indeed be suitable for chemotherapeutic studies relevant to man. In addition, the more rapid disappearance of dead M. leprae from the rat tissues may facilitate the study of treatment regimens designed to eradicate persisting viable organisms.

Dapsone chemotherapy of Mycobacterium leprae infection of the neonatally thymectomized Lewis rat

In order to learn whether the neonatally thymectomized Lewis rat (NTLR) infected with Mycobacterium leprae could serve as a model for chemotherapeutic studies in a situation resembling that found in human lepromatous leprosy, NTLR inoculated with M. leprae either locally or intravenously 9 to 16 months earlier were treated for from 1.5 to 8.5 months with dapsone (4,4´-diaminodiphenylsulfone, DDS) incorporated in the rat chow in the concentration providing the minimal inhibitory concentration of the drug for M. leprae and in the 100-fold larger concentration. NTLR were killed at intervals; the M. leprae were counted and passed to mice. Treatment with the smaller dosage of dapsone neither killed M. leprae nor reduced the number of organisms in the bacterial populations, whereas treatment with the larger dosage both killed M. leprae and reduced their numbers. The rate at which the organisms were killed (i.e., rendered noninfective for mice) was much the same as that in patients treated with dapsone in comparable dosage. The dead organisms were removed from the rat tissues at a faster rate than encountered in patients. The NTLR may indeed be suitable for chemotherapeutic studies relevant to man. In addition, the more rapid diappearance of dead M. leprae from the rat tissues may facilitate the study of treatment regimens designed to eradicate persisting viable organisms.