Alteration of cytokine production in follicular cystic ovaries induced in mice by neonatal estradiol injection

PROBLEM: Neonatal estradiol injections in mice lead to follicular cystic ovaries that are similar to ovaries in patients with polycystic ovarian syndrome (PCOS). The present study examined ovarian cytokine production following neonatal estradiol injection. METHOD OF STUDY: Female (C3H,HeJ x 129/HeJ)F1 mice were injected daily with 20 microg 17beta-estradiol from 0-3 days postpartum. At intervals, animals were sacrificed to determine ovarian architecture, circulating levels of estradiol, ovarian and peritoneal macrophage cytokine production, and ovarian P450 aromatase enzyme mRNA levels. RESULTS: Similar to PCOS, our results show that neonatally estradiol-injected mice have lower levels of circulating estrogen that are correlated with decreased mRNA levels of P450 aromatase enzyme. Our data also show that follicular cystic ovaries have increased tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 production. This increase in TNF-alpha and IL-6 production is also observed in peritoneal macrophages of estradiol-injected mice. CONCLUSION: The present study showed that neonatal estrogen injection in mice has an overall systemic effect on cytokine production. We speculate that increased cytokine production may alter certain important steps in follicular maturation, ultimately contributing to ovarian dysfunction.     

Episomal and stable expression of the luciferase reporter gene for quantifying Leishmania spp. infections in macrophages and in animal models

We have expressed the reporter firefly luciferase gene (LUC) in Leishmania donovani and Leishmania major either as part of episomal vectors or integrated into the parasite genome under the control of their respective ribosomal promoter regions. An excellent linear correlation between parasite number and luciferase activity was observed with all the transfectants. LUC-expressing recombinant parasites were useful to monitor Leishmania spp. infections in macrophages or in animal models. For prolonged growth in absence of drug selection, such as within animal models, quantitation of parasites is more reliable when the reporter gene LUC is stably integrated in the parasite genome. These recombinant strains should be useful tools to monitor Leishmania growth under a number of conditions.     

Long-term inhibition of intimal hyperplasia using vascular photodynamic therapy in balloon-injured carotid arteries

Flexible treatments for intimal hyperplasia after angioplasty are still needed. The aim of this study was to demonstrate the long-term effects of vascular photodynamic therapy with talaporfin sodium on intimal hyperplasia following interventional injury. Intimal hyperplasia was induced by balloon distension injury to the carotid artery in 31 rabbits. Talaporfin, 5.0 mg/kg, was delivered systemically immediately after balloon injury. The injury site was irradiated with a diode laser light of wavelength 664 nm using a fluence of 50 J/cm² after 30 min. At day 3 and weeks 3, 6, 9, 15, and 25 after photodynamic therapy, the treated artery of each rabbit was excised and examined immunohistochemically. Thirty minutes after talaporfin administration, drug fluorescence was found only in the balloon-injured carotid artery wall. At 3 days, no smooth muscle cells were seen in the media of the photodynamic therapy-treated arterial segments. Intimal hyperplasia developed progressively in the balloon-injured and untreated segments; however, in the segments treated with photodynamic therapy, intimal hyperplasia was markedly suppressed until 25 weeks and the media was repopulated by smooth muscle cells without macrophages. Vascular photodynamic therapy with talaporfin may be used to inhibit restenosis after vascular intervention. An erratum to this article is available at .     

Pre-exposure of murine macrophages to lipopolysaccharide inhibits the induction of nitric oxide synthase and reduces leishmanicidal activity

Murine macrophages produce nitric oxide (NO) from L-arginine on stimulation with lipopolysaccharide (LPS), alone or with interferon-γ (IFN-γ). The effect of incubation of macrophages with low concentrations of LPS on NO synthesis on subsequent stimulation was investigated, using a murine macrophage cell line, J774, and peritoneal macrophages from CBA mice. Cells which had been incubated with LPS produced significantly lower amounts of NO, and expressed lower levels of NO synthase activity, following stimulation with IFN-γ and LPS, or with a high concentration of LPS. This effect was not reversed by tumor necrosis factor-α. The ability of CBA macrophages to kill the intracellular parasite Leishmania major was markedly reduced by pre-incubation with LPS. Reduced NO production by macrophages previously exposed to LPS is a manifestation of endotoxin tolerance, and may represent an important means of regulation of NO synthesis and thus a survival mechanism for intracellular parasites.     

Morphological features of nerve terminal degeneration as part of the remodeling process in the motor endplate in adult muscles

With the exception of signs of retraction and withdrawal, there have been few morphological data concerning degenerated neural profiles in adult motor endplates. Here, investigation into the ultrastructure of the soleus motor endplates of adult rats (4 months old) turned up particular axonal degeneration in approximately 3% of the subjects. These axons occur as synaptic debris in the synaptic matrix of the motor endplate, adjacent to thin processes of the perisynaptic cells occupying the outer most layer of the motor endplate and were devoid of basal lamina. They often possessed dense-cored vesicles (50-80 nm). Axonal debris released from Schwann cell processes occurred during the period of acute sciatic neurectomy, when nerve terminals progressively disrupted within the motor endplate associated Schwann cells. Finally, immunohistochemical staining for antibodies to label macrophages (ED1 or ED2) has shown that nerve fiber-associated macrophages are located near the motor endplate. The results suggest that during the course of endplate remodeling, a few parts of the terminal branches are disposed of through spontaneous collapse, subsequent release from the Schwann cell investment, and eventual ingestion by macrophages in the perisynaptic space.     

Histamine: Its novel role as an endogenous regulator of Con A – dependent T cell proliferation

Objective and design:The roles of histamine formed by the macrophage – T lymphocyte system were evaluated in the regulation of lymphocyte proliferation using mice lacking histamine receptors. Methods:Mice deficient in histamine type 1 (H1R), type 2 (H2R) or both receptors were employed to estimate possible intervention of the receptors in the histamine-dependent lymphocyte proliferation. Results:Histamine was produced de novo by spleen cells. Con A-dependent T cell proliferation decreased when histamine produced in the culture was degraded by the addition of histaminase. The H2R-deficient mice also showed a significant decrease in the Con A-dependent T cell proliferation, whereas it was not modulated in the H1R-deleted mice. Consistent with the reduction in T cell proliferation, there was a significant down-regulation of the production of IL-2, a T cell growth factor, in the H2R-deficient mice. Con A-dependent IL-2 synthesis was abrogated by the addition of histaminase. Conclusions:Con A-dependent T cell proliferation is (up)regulated by histamine produced de novo through the H2R, suggesting that histamine is a newly found regulator of T cell proliferation.Received 18 October 2003; returned for revision 17 December 2003; accepted by M. J. Parnham 6 February 2004     

MacrophAging: a cellular and molecular review

Aging is associated with the deterioration of several physiological functions, which leads to aged-related pathologies and, ultimately, to death. The immune system is affected by aging, causing an increased susceptibility to infections and mortality, as well as a major incidence of immune diseases and cancer in the elderly. Because macrophages are an essential component of both innate and adaptive immunity, altered function of these phagocytic cells with aging may play a key role in immunosenescence. Here we summarize data about the effects of aging on macrophages and we discuss the molecular events that could be involved in this process.     

Immunohistochemical analysis of markers for different macrophage phenotypes and their use for a forensic wound age estimation

A total of 117 vital skin wounds (post infliction intervals between a few seconds and 7 months), 20 postmortem wounds and skin specimens with beginning or advanced signs of putrefaction were investigated. Different markers for macrophage maturation (27 E 10, RM 3/1, 25 F 9, G 16/1) were analyzed by immunohistochemistry. The early stage inflammation marker 27 E 10 stained macrophages, but also monocytes and neutrophilic granulocytes localized in blood vessels or bleeding induced postmortem and therefore provided no further information for a forensic wound age estimation in comparison to the routine histological detection of macrophages. The antigens recognized by the RM 3/1- (intermediate stage inflammation marker) and 25 F 9-antibodies (late stage inflammation marker) were expressed exclusively by histiocytes and inflammatory cells that had migrated from the blood vessels as part of the acute inflammatory response associated with an intravital reaction. The morphometrical analysis revealed positive results (defined as at least a two-fold increase in number in 2 or more microscope fields when compared to the maximum value of histiocytes found in uninjured skin) for the RM 3/1- or 25 F 9-antibody earliest in wounds aged 7 or 11 days, respectively. Similarly to the 25 F 9-antibody, the chronic stage inflammation marker (G 16/1) reacted with a macrophage subpopulation first detectable 12 days after wounding but showed positive results in a comparably reduced percentage of cases. On the other hand, this marker did not stain a relevant number of resident macrophages thus facilitating the evaluation of the specimens. The markers 27 E 10, RM 3/1 and 25 F 9 are also useful for the evaluation of slightly - even though the staining intensity was considerably reduced - but not advanced putrefied skin. Therefore, the immunohistochemical analysis of the corresponding antigens can possibly contribute to an age estimation of wounds with advanced post infliction intervals obtained from corpses with longer - but limited - postmortem intervals.

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Zusammenfassung Insgesamt wurden 117 vitale Hautwunden (Überlebenszeit wenige Sekunden bis 7 Monate), 20 postmortal gesetzte Verletzungen sowie Haut mit leichten bzw. fortgeschrittenen Fäulnisveränderungen untersucht und verschiedene Marker der Makrophagen-Differenzierung (27 E 10, RM 3/l, 25 F 9 und G 16/1) analysiert. Der early stage inflammation marker 27 E 10 färbte neben Makrophagen auch Monozyten und neutrophile Granulozyten, die innerhalb von Blutgefäßen bzw. in postmortal gesetzten Blutungen lokalisiert waren und liefert somit keine Informationen zum Wundalter, die über die Möglichkeiten des Routine-histologischen Nachweises von Makrophagen hinausgingen. Die von den Antikörpern RM 3/1 (intermediate stage inflammation marker) und 25 F 9 (late stage inflammation marker) erkannten Antigene wurden ausschließlich von Histiozyten und reaktiv eingewanderten Makrophagen exprimiert. Die morphometrische Analyse ergab positive Ergebnisse (definiert als ein mindestens zweifacher Anstieg der Zellzahl in zwei oder mehr Gesichtsfeldern verglichen mit der maximal feststellbaren Zahl an Histiozyten in unverletzter Haut) bei Verwendung der Antikörper RM 3/1 bzw. 25 F 9 frühestens 7 bzw. 11 Tage nach Wundsetzung. Ab 12 Tagen Wundalter reagierte der chronic stage inflammation marker G 16/1 erstmals positiv. Das Antigen ließ sich insgesamt allerdings in einem geringeren Prozentsatz der untersuchten Wunden darstellen. Vorteilhaft ist jedoch das Fehlen einer relevanten Expression durch Histiozyten, wodurch die Auswertung der Präparate erleichtert wird. Die entsprechenden Antigene lassen sich zudem in leicht - wenn auch in einer deutlich geringeren Färbeintensität -, aber nicht forgeschritten fäulnisveränderter Haut nachweisen, so daß deren immunhistochemische Darstellung gegebensfalls auch zur Beurteilung von länger überlebten Verletzungen an Leichen mit etwas fortgeschrittener Liegezeit herangezogen werden kann.

     

Immunohistochemical dynamics of leukotoxin (9.10-eooxv-12-octadecenoic acid) in lungs of rats

This paper investigates the immunohistochemical dynamics of leukotoxin (9,10-epoxy-12-octadecenoic acid, LTx) in the lungs of rats exposed to hyperoxia with or without paraquat. The rats were treated with 100% oxygen or ambient air for 24. 48, 72 and 96 h in the presence or absence of a low or high dose paraquat (1,1-di-methyl-4,4-bipyridinium, PQ) injection. Immunostaining for LTx demonstrated positive reactions in the neutrophils that showed a progressive increase in intensity of staining with time in all groups exposed to 100% oxygen and in the group with high dose PQ, but the positive findings were weak in the group injected with low dose PQ only. We found the positive immunostaining reaction not only in neutrophils but also in alveolar macrophages. This indicates that LTx is produced by alveolar macrophages as well as by neutrophils depending on the treatment period under hyperoxic conditions, suggesting that LTx is an important chemical mediator in pulmonary diseases.     

脾切除对小鼠腹腔巨噬细胞数量和功能的影响

目的：研究脾切除对小鼠腹腔巨噬细胞数量和功能活性的影响。方法：术后4周获取腹腔常居巨噬细胞和炎症巨噬细胞,检测其数量、吞噬功能和一氧化氨水平。结果：与正常对照组和假手术组比较,脾切除组小鼠的2种巨噬细胞不仅数量明显减少,而且它们的吞噬功能和一氧化氮水平呈明显下降。结论：提示脾切除造成的单核吞噬细胞继发性炎症反应的捐伤和巨噬细胞功能活性的降低,可能是脾切除后(凶险性)感染的重要因素。