Self-Maintaining Gut Macrophages Are Essential for Intestinal Homeostasis.

Macrophages are highly heterogeneous tissue-resident immune cells that perform a variety of tissue-supportive functions. The current paradigm dictates that intestinal macrophages are continuously replaced by incoming monocytes that acquire a pro-inflammatory or tissue-protective signature. Here, we identify a self-maintaining population of macrophages that arise from both embryonic precursors and adult bone marrow-derived monocytes and persists throughout adulthood. Gene expression and imaging studies of self-maintaining macrophages revealed distinct transcriptional profiles that reflect their unique localization (i.e., closely positioned to blood vessels, submucosal and myenteric plexus, Paneth cells, and Peyer's patches). Depletion of self-maintaining macrophages resulted in morphological abnormalities in the submucosal vasculature and loss of enteric neurons, leading to vascular leakage, impaired secretion, and reduced intestinal motility. These results provide critical insights in intestinal macrophage heterogeneity and demonstrate the strategic role of self-maintaining macrophages in gut homeostasis and intestinal physiology.

Macrophages in the gut: Masters in multitasking.

The gastrointestinal tract has the important task of absorbing nutrients, a complex process that requires an intact barrier allowing the passage of nutrients but that simultaneously protects the host against invading microorganisms. To maintain and regulate intestinal homeostasis, the gut is equipped with one of the largest populations of macrophages in the body. Here, we will discuss our current understanding of intestinal macrophage heterogeneity and describe their main functions in the different anatomical niches of the gut during steady state. In addition, their role in inflammatory conditions such as infection, inflammatory bowel disease, and postoperative ileus are discussed, highlighting the roles of macrophages in immune defense. To conclude, we describe the interaction between macrophages and the enteric nervous system during development and adulthood and highlight their contribution to neurodegeneration in the context of aging and diabetes.

Dedicated macrophages organize and maintain the enteric nervous system.

Correct development and maturation of the enteric nervous system (ENS) is critical for survival1. At birth, the ENS is immature and requires considerable refinement to exert its functions in adulthood2. Here we demonstrate that resident macrophages of the muscularis externa (MMϕ) refine the ENS early in life by pruning synapses and phagocytosing enteric neurons. Depletion of MMϕ before weaning disrupts this process and results in abnormal intestinal transit. After weaning, MMϕ continue to interact closely with the ENS and acquire a neurosupportive phenotype. The latter is instructed by transforming growth factor-ß produced by the ENS; depletion of the ENS and disruption of transforming growth factor-ß signalling result in a decrease in neuron-associated MMϕ associated with loss of enteric neurons and altered intestinal transit. These findings introduce a new reciprocal cell-cell communication responsible for maintenance of the ENS and indicate that the ENS, similarly to the brain, is shaped and maintained by a dedicated population of resident macrophages that adapts its phenotype and transcriptome to the timely needs of the ENS niche.

Local immune response to food antigens drives meal-induced abdominal pain.

Up to 20% of people worldwide develop gastrointestinal symptoms following a meal1, leading to decreased quality of life, substantial morbidity and high medical costs. Although the interest of both the scientific and lay communities in this issue has increased markedly in recent years, with the worldwide introduction of gluten-free and other diets, the underlying mechanisms of food-induced abdominal complaints remain largely unknown. Here we show that a bacterial infection and bacterial toxins can trigger an immune response that leads to the production of dietary-antigen-specific IgE antibodies in mice, which are limited to the intestine. Following subsequent oral ingestion of the respective dietary antigen, an IgE- and mast-cell-dependent mechanism induced increased visceral pain. This aberrant pain signalling resulted from histamine receptor H1-mediated sensitization of visceral afferents. Moreover, injection of food antigens (gluten, wheat, soy and milk) into the rectosigmoid mucosa of patients with irritable bowel syndrome induced local oedema and mast cell activation. Our results identify and characterize a peripheral mechanism that underlies food-induced abdominal pain, thereby creating new possibilities for the treatment of irritable bowel syndrome and related abdominal pain disorders.

Pragmatic computerised perfusion diagnostics for non-convulsive status epilepticus: a prospective observational study.

BACKGROUND: Non-convulsive status epilepticus (NCSE) is a time-dependent neurological disorder often misdiagnosed in the emergency setting. Electroencephalography (EEG) is often not available on a 24/7 basis, and Salzburg criteria may at times miss the diagnosis. Here, we tested the accuracy of hyperperfusion on CT perfusion imaging (CTP) in the identification of NCSE against Salzburg criteria, to define its potential role in a pragmatic diagnostic workflow. METHODS: We enrolled consecutive patients with suspected acute seizure or seizure disorder undergoing brain imaging with CTP and EEG from January 2021 to March 2023. EEG recordings, Salzburg criteria and CTP hyperperfusion were rated and adjudicated by two independent experts blinded to patient status. A reference standard including all clinical, lab, imaging, EEG and therapeutic data was used to adjudicate NCSE diagnosis. Sensitivity, specificity, diagnostic accuracy, positive and negative predictive values (NPV) were calculated for CTP hyperperfusion and Salzburg criteria versus NCSE adjudicated according to reference standard. RESULTS: Seventy-seven patients were enrolled. Among 21 NCSE cases, 17 were adjudicated according to Salzburg criteria (81%) and 4 received NCSE diagnosis according to reference standard. Agreement between EEG and CTP emerged in 16/21 NCSE cases, reaching sublobar level in 37.5% of cases. Receiver operator curve analysis suggested good accuracy for CTP hyperperfusion for the diagnosis of NCSE (AUROC 0.79, 95% CI 0.69 to 0.89). CTP hyperperfusion had a high NPV for NCSE (NPV 0.97, 95% CI 0.86 to 1). CONCLUSION: CTP hyperperfusion may be implemented in the emergency fast-track to rule out NCSE, given very high NPV. Further validation studies are needed to evaluate CTP application in real-world setting for NCSE codes.

Are Retinal Detachments Becoming More Frequent? Data from 2 University Eye Clinics and Literature Review. / Werden Netzhautablösungen häufiger? Daten von 2 Universitätsaugenkliniken und Literaturübersicht.

There have been marked increases in the numbers of patients with retinal detachments at individual centres in recent years and this is supported by the subjective impression of many experts. We therefore surveyed the literature on changes in the incidence of retinal detachments worldwide. This revealed quite significant methodological differences between the studies, so that it was difficult to achieve a conclusive comparison of the development of the incidence of retinal detachment. Despite these limitations, all data from recent studies suggest an increase in the number of retinal detachments. The incidence of retinal detachment in the western world currently seems to be more than 20 cases per 100,000 person-years, which is significantly higher than described in earlier decades. It can be assumed that an increase in the number of individuals with myopia, a demographic increase in patients of the typical age group for retinal detachment and an increasing number of cataract surgeries, especially in younger patients, are responsible for the rising incidence of retinal detachment.

[Prevalence and geographical distribution of bovine sexually transmitted diseases in the province of Formosa, Argentina]. / Prevalencia y distribución geográfica de las enfermedades de transmisión sexual de los bovinos en la provincia de Formosa, Argentina.

Bovine genital campylobacteriosis (BGC) and bovine trichomonosis (BT) are sexually transmitted diseases (STDs) that affect bovine breeding herds, decreasing their reproductive efficiency. The objective of this work was to estimate the prevalence of these diseases and their temporal-spatial distribution in the province of Formosa, Argentina. The cross-sectional study conducted between 2018 and 2021 included a total of 15,571 bulls, inter-herd prevalence being 29.62% and 17.23% for BGC and BT, respectively. The prevalence of positive animals was 2.05% for BGC and 0.43% for BT. The temporal-spatial analysis of BGC showed two distinct spatial groupings, one group had a low risk of contracting the disease (RR = 0.13; p < 0.001; 2018-2021) while the other group had a high risk (RR = 2.84; p < 0.001; 2020-2021). BT had a high-risk group for the disease (RR = 35.24; p < 0.001; 2019). This study shows that STDs are endemic in the region, providing updated and valuable information as a tool for the health management of these diseases.

Percutaneous Chevron Osteotomy: A Prospective Randomized Controlled Trial.

Introduction: Minimally invasive surgical techniques for hallux valgus have gained popularity, showing good results characterized by smaller postoperative scars, less pain, lower infection risk, and fewer wound complications. Given the lack of evidence available in our country regarding this subject, especially about this type of surgical technique, our paper aims to compare open and MIS approaches for chevron osteotomy. We evaluated the outcome and complications after 12 months. Materials and Methods: We undertook a prospective, randomized, controlled, single-center study between October 2017 and December 2020. The patients were randomized into two groups: one group that received percutaneous chevron osteotomy (MIS), and the other, open chevron osteotomy (OC). For clinical assessment, we determined the function and the level of pain using the Visual Analogue Scale (VAS) and The American Orthopaedic Foot and Ankle Surgery score (AOFAS). The VAS scale was measured before the surgical procedure, at discharge, and at 3 weeks, 6 weeks, 6 months, and 12 months after surgery. The AOFAS score was calculated preoperatively and after 6 months. The hallux angle (HVA) and intramedullary angle (IMA) were measured preoperatively, and at 6 weeks, 6 months and 12 months. Results: We included 26 cases in the open chevron osteotomy group (24 female, 2 male) and 24 in the MIS group (24 female, 0 male). Both groups demonstrated improvements regarding the IMA and HVA at the last follow-up without any significant differences between the groups at the final assessment. The VAS showed significantly better post-operative results for the MIS group at discharge (p < 0.001) and 3 weeks (p < 0.001), 6 weeks (p < 0.001), and 6 months (p = 0.004) post-surgery. The AOFAS showed no significant differences either before or after surgery. Four cases with screw prominence were reported, three of which belonged to the MIS group. Only one case with metatarsalgia was found in the OC group. Conclusions: This paper demonstrates that minimally invasive chevron osteotomy has comparable results with open chevron osteotomy, even though surgical time and radiological exposure are significantly longer. More studies are required to evaluate the complications and the risk of recurrences.

Niche-specific functional heterogeneity of intestinal resident macrophages.

Intestinal resident macrophages are at the front line of host defence at the mucosal barrier within the gastrointestinal tract and have long been known to play a crucial role in the response to food antigens and bacteria that are able to penetrate the mucosal barrier. However, recent advances in single-cell RNA sequencing technology have revealed that resident macrophages throughout the gut are functionally specialised to carry out specific roles in the niche they occupy, leading to an unprecedented understanding of the heterogeneity and potential biological functions of these cells. This review aims to integrate these novel findings with long-standing knowledge, to provide an updated overview on our understanding of macrophage function in the gastrointestinal tract and to speculate on the role of specialised subsets in the context of homoeostasis and disease.

Comparison of PCR-based methods for the detection of Babesia caballi and Theileria equi in field samples collected in Central Italy.

Equine piroplasmosis (EP) is a disease of equids caused by Theileria equi and Babesia caballi, members of the order Piroplasmida, transmitted by several species of ticks. As the disease is endemic in many countries, a clinical examination or a serological test are required prior to movement of horses to prove freedom from infection and to avoid the introduction of EP with its sanitary and economic impact, especially in areas where it is absent. Currently, numerous diagnostic PCR protocols are available, some of which are recommended by the World Organisation for Animal Health (OIE). In order to adopt this diagnostic method, the Italian National Reference Centre for Equine Diseases (NRC-ED) conducted a preliminary comparison between an end-point PCR, nested PCR, real-time PCR, and commercial real-time PCR, for the detection of T. equi and B. caballi, respectively. One hundred and three field samples, collected during spring-summer 2013 in Latium and Tuscany regions, were employed for the study, and results discordant between detection assays were confirmed by sequencing. The reference assay was defined as that showing the highest sensitivity, and the relative sensitivity (rSe) and specificity (rSp) of the other methods were estimated referring to this assay. Agreement between methods was estimated by calculating the concordance between each pair of methods. Although no statistical differences were detected among PCR-based methods, the non-commercial real-time PCR assays seemed to be the most suitable for detection of T. equi and B. caballi, respectively. An important advantage of direct PCR detection of the pathogen, in comparison to indirect detection using serological methods, is that it allows specific treatment against the causative pathogen species responsible of the infection as well as for the definition of the infectious status of an animal for international movement.

Thermal Laser Monotherapy for Extrafoveal Polypoidal Choroidal Vasculopathy.

INTRODUCTION: Polypoidal choroidal vasculopathy (PCV) is a vascular disease of the choroid. Diagnosis is mainly based on polypoidal aneurysm-like lesions seen in indocyanine green (ICG) angiography. Various therapeutic options have been proposed. METHODS: Outcomes of 10 cases with extrafoveal PCV and consecutive macular edema treated with thermal laser are reported. Diagnosis of PCV was confirmed by ICG angiography. RESULTS: Upon successful occlusion of the polyps in 10 eyes after thermal laser treatment demonstrated in ICG angiography, a regression of central foveal edema was seen in optical coherence tomography and color fundus photography. Visual acuity improved from logMAR 0.8 to logMAR 0.3. Follow-up ranged from 4 months to 15 years, with a median of 1 year. Two eyes had a recurrence of exudative maculopathy 5 and 7 years after laser treatment, respectively. CONCLUSION: A careful differentiation between various subforms of exudative maculopathy using fluorescein and ICG angiography can identify certain selected patients with extrafoveal PCV, for whom thermal laser monotherapy can be a therapeutic option.

FLT3-ITD DNA and mRNA levels in AML do not correlate with CD7, CD33 and CD123 expression.

INTRODUCTION: FLT3 internal tandem duplication (ITD) mutations are found in around 25% of all acute myeloid leukaemia (AML) cases and is associated with shorter disease-free and overall survival. Previous reports have shown that FLT3-ITD induces a specific phenotype in leukemic blasts, which is characterized by high levels of CD33 and CD123, and that expression of CD33 and CD123 is directly influenced by the DNA FLT3-ITD/wild-type FLT3 allelic ratio (AR). METHODS: A total of 42 FLT3-ITD and 104 FLT3-ITD-negative AML patients were analysed. Immunophenotyping data were used to calculate antigen expression levels as the ratio between the geometric mean fluorescence intensities (MFIs) of leukemic blasts and MFIs of negative lymphocyte populations. FLT3-ITD-DNA and RNA analysis was performed, under the same conditions, by capillary electrophoresis. RESULTS: Compared with the control group, the FLT3-ITD cohort presented significantly higher CD7, CD33 and CD123 levels. In order to assess the impact of FLT3-ITD abundance on antigen expression, the patients were grouped for each parameter into two cohorts using the following threshold values: (a) 0.5 for the AR, according to current AML guidelines; (b) 0.7 for the FLT3-ITD/FLT3-WT mRNA ratio (RR); and (c) 1.3 for the FLT3-ITD RR/AR ratio. We found higher values of CD33 for RR/AR ≥1.3, and no other statistical differences between CD7, CD33 and CD123 levels of the other FLT3-ITD groups. In terms of correlations between MFI values and FLT3-ITD parameters, we only observed a moderate interdependence between CD33 MFI and the RR/AR ratio, and a weak negative correlation between CD123 MFI and AR. CONCLUSION: FLT3-ITD mutations induce a specific antigen profile in AML blasts, and our data do not onfirm previous reports of FLT3-ITD AR influencing both CD33 and CD123 expression.

Preoperative administration of the 5-HT4 receptor agonist prucalopride reduces intestinal inflammation and shortens postoperative ileus via cholinergic enteric neurons.

OBJECTIVES: Vagus nerve stimulation (VNS), most likely via enteric neurons, prevents postoperative ileus (POI) by reducing activation of alpha7 nicotinic receptor (α7nAChR) positive muscularis macrophages (mMφ) and dampening surgery-induced intestinal inflammation. Here, we evaluated if 5-HT4 receptor (5-HT4R) agonist prucalopride can mimic this effect in mice and human. DESIGN: Using Ca2+ imaging, the effect of electrical field stimulation (EFS) and prucalopride was evaluated in situ on mMφ activation evoked by ATP in jejunal muscularis tissue. Next, preoperative and postoperative administration of prucalopride (1-5 mg/kg) was compared with that of preoperative VNS in a model of POI in wild-type and α7nAChR knockout mice. Finally, in a pilot study, patients undergoing a Whipple procedure were preoperatively treated with prucalopride (n=10), abdominal VNS (n=10) or sham/placebo (n=10) to evaluate the effect on intestinal inflammation and clinical recovery of POI. RESULTS: EFS reduced the ATP-induced Ca2+ response of mMφ, an effect that was dampened by neurotoxins tetrodotoxin and ω-conotoxin and mimicked by prucalopride. In vivo, prucalopride administered before, but not after abdominal surgery reduced intestinal inflammation and prevented POI in wild-type, but not in α7nAChR knockout mice. In humans, preoperative administration of prucalopride, but not of VNS, decreased Il6 and Il8 expression in the muscularis externa and improved clinical recovery. CONCLUSION: Enteric neurons dampen mMφ activation, an effect mimicked by prucalopride. Preoperative, but not postoperative treatment with prucalopride prevents intestinal inflammation and shortens POI in both mice and human, indicating that preoperative administration of 5-HT4R agonists should be further evaluated as a treatment of POI. TRIAL REGISTRATION NUMBER: NCT02425774.

Comparison of the enzymatic efficiency of Liberase TM and tumor dissociation enzyme: effect on the viability of cells digested from fresh and cryopreserved human ovarian cortex.

BACKGROUND: The aim of this study was to examine the effectiveness of Tumor Dissociation Enzyme (TDE) on the viability of follicles after digestion of fresh and cryopreserved ovarian cortex fragments (OCFs). METHODS: Fresh and thawed OCF from 14 patients (29 ± 6 years), sized 20 to 210 mm3 were randomly distributed into four treatment groups and digested with 16% TDE or 0.05 mg/ml Liberase TM: Group 1, frozen OCF digested with TDE; Group 2, frozen OCF digested with LiberaseTM; Group 3, fresh OCF digested with TDE; and Group 4, fresh OCF digested with Liberase TM. Evaluation of follicle viability was performed under light microscope after staining with Neutral red. For visualization of viable and dead cells under a confocal laser scanning microscope, the follicles were stained with Calcein AM and ethidium homodimer-1. RESULTS: The results showed that the number of retrieved follicles was significantly higher (990 vs 487; P < 0.01) in the TDE-treatment group compared to the Liberase TM-group. The presence of intense neutral red stained follicles was significantly higher in Group 1 and Group 3 compared to Group 2 and Group 4 (70.3% ± +/- 6.22 vs 53,1% ± 2.03 and 94.2% ± 6.6 vs 79.1% ± 2.1; P < 0.01). The percentage of Calcein AM stained follicles of class V1 was significantly higher in Group 1 and Group 3 compared to Group 2 and Group 4 (95.97% ± 7.8 vs 87.87% ± 2.4; 97.1% ± 6.8 vs 91.3% ± 2.3; P < 0.01). CONCLUSION: The enzymatic digestion of ovarian cortex with TDE provides recovery of a higher number of healthy preantral follicles in contrast to earlier described Liberase TM procedure.

Construction of human artificial ovary from cryopreserved ovarian tissue: Appearance of apoptosis and necrosis after enzymatic isolation of follicles.

Earlier it was shown that number of retrieved follicles was significantly higher in Tumor Dissociation Enzyme (TDE)-treatment group compare to standard Liberase TM-group. The aim of our present investigations was to examine the effect of TDE on appearance of apoptosis and necrosis in follicles and stromal cells after digesting of cryopreserved ovarian cortex. Fresh and frozen ovarian cortex fragments (OCF) from 14 patients (29 ±â€¯6 years old), sized 20-210 mm3 were randomly distributed into four treatment groups and digested with 16% TDE or 0.05 mg/ml Liberase TM: Group 1 frozen OCF digested with TDE; Group 2 frozen OCF digested with LiberaseTM; Group 3 fresh OCF digested with TDE; Group 4 fresh OCF digested with Liberase TM. To differentiate the live, early apoptotic, late apoptotic and necrotic cells in digested ovarian cortex suspension, a flow cytometric apoptosis/necrosis assay with FITC Annexin V Apoptosis Detection Kit and with 7-AAD was performed. Most of fresh (not frozen) cells digested with TDE or Liberase TM (95 ±â€¯2.4% vs. 90.4 ±â€¯3.1%, respectively) as well as in frozen ovarian cortex digested with TDE or Liberase TM (93.1 ±â€¯3.4% vs. 89.7 ±â€¯4.4%, respectively) has located in Q3 quadrant and these cells both negative to 7-AAD and Annexin V were considered as viable. It was established that both types of enzymatic treatment applying to fresh as well as to frozen ovarian cortex resulted to high rate of viable cells (Group 1: 93.8 ±â€¯3.4%; Group 2: 91.8 ±â€¯6.0%; Group 3: 90.5 ±â€¯6.9%; Group 4: 87.3 ±â€¯2.3%) and are non significantly different (P > 0.1) between all treatment groups. The amount of early apoptotic (Group 1: 3.5 ±â€¯1.6%; Group 2: 4.4 ±â€¯1.6%; Group 3: 1.6 ±â€¯1.1%; Group 4: 2.4 ±â€¯1.5%), late apoptotic (Group 1: 2.7 ±â€¯2.4%; Group 2: 44.0 ±â€¯1.9%; Group 3: 3.1 ±â€¯1.1%; Group 4: 2.8 ±â€¯0.7% and necrotic (Group 1: 0.9% ±â€¯0.1%; Group 2: 2.9 ±â€¯0.8%; Group 3: 3.4 ±â€¯4.5%; Group 4: 1.1 ±â€¯0.6%) cells was low and was not significantly different in all treatment groups (P > 0.1). It was concluded that the use of Tumor Dissociation Enzyme, effectiveness of which is higher than Liberase TM, does not lead to increasing of apoptosis and necrosis in follicles and stromal cells after enzymatic digesting of cryopreserved ovarian cortex.

Antimicrobial and Anti-Proliferative Effects of Skin Mucus Derived from Dasyatis pastinaca (Linnaeus, 1758).

Resistance to chemotherapy occurs in various diseases (i.e., cancer and infection), and for this reason, both are very difficult to treat. Therefore, novel antimicrobial and chemotherapic drugs are needed for effective antibiotic therapy. The aim of the present study was to assess the antimicrobial and anti-proliferative effects of skin mucus derived from Dasyatis pastinaca (Linnaeus, 1758). Our results showed that skin mucus exhibited a significant and specific antibacterial activity against Gram-negative bacteria but not against Gram-positive bacteria. Furthermore, we also observed a significant antifungal activity against some strains of Candida spp. Concerning anti-proliferative activity, we showed that fish mucus was specifically toxic for acute leukemia cells (HL60) with an inhibition of proliferation in a dose dependent manner (about 52% at 1000 µg/mL of fish skin mucous, FSM). Moreover, we did not observe effects in healthy cells, in neuroblastoma cells (SH-SY5Y), and multiple myeloma cell lines (MM1, U266). Finally, it exhibited strong expression and activity of chitinase which may be responsible, at least in part, for the aforementioned results.

Neuroactive molecules and growth factors modulate cytoskeletal protein expression during astroglial cell proliferation and differentiation in culture.

Steroid hormones and neurotrophic factors regulate astroglial cell survival, proliferation, and differentiation in culture. The present study examines the interaction between glucocorticoids and growth factors (GFs) on cytoskeletal proteins and extracellular signal-regulated kinase 2 (ERK2) expression in stressed astroglial cultures at 25 days in vitro, according to the following experimental condition. Pretreatment with basic fibroblast growth factor alone or in combination with dexamethasone 10(-9) M for 48 hr induced an enhancement of glial fibrillary acidic protein, vimetin, and ERK2 expression. Treatment with "progression" GFs alone and in the last 12 hr significantly increased the above-mentioned markers' expression. The present study shows that glucocorticoids may cooperate with GFs or may abrogate their effects, depending on the experimental culture conditions used as well as the exposure time and the types of GFs added. Our findings provide evidence of interactive dialogue between GFs and neurosteroids in cultured astrocytes. This may have implications in the therapeutic approach to neurologic disorders associated with astrogliosis.

Modulation of extracellular signal-related kinase, cyclin D1, glial fibrillary acidic protein, and vimentin expression in estradiol-pretreated astrocyte cultures treated with competence and progression growth factors.

The present study seeks to elucidate the interactions between the "competence" growth factor basic fibroblast growth factor (bFGF) and/or estrogen 17ß-estradiol and the "progression" growth factors epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), and insulin (INS) on DNA labeling and also cyclin D1, extracellular signal-related kinase 1/2 (ERK1/2), glial fibrillary acidic protein (GFAP), and vimentin expression in astroglial cultures under different experimental conditions. Pretreatment for 24 hr with bFGF and subsequent exposure for 36 hr to estradiol (E2 ) and EGF, IGF-I, or INS stimulated DNA labeling in the last 12 hr, especially when the cultures were treated with progression growth factors. bFGF pretreatment and subsequent treatment with E2 for 36 hr stimulated DNA labeling. The 36-hr E2 treatment alone did not significantly decrease DNA labeling, but contemporary addition of E2 with two or three growth factors stimulated DNA labeling remarkably. When E2 was coadded with growth factors, a significantly increased DNA labeling was observed, demonstrating an astroglial synergistic mitogenic effect evoked by contemporary treatment with growth factors in the presence of estrogens. Cyclin D1 expression was markedly increased when astrocyte cultures were pretreated for 36 hr with E2 and subsequently treated with two or three competence and progression growth factors. A highly significant increase of ERK1/2 expression was observed after all the treatments (EGF, bFGF, INS, IGF-I alone or in combination with two or three growth factors). GFAP and vimentin expression was markedly increased when the cultures were treated with two or three growth factors. In conclusion, our data demonstrate estradiol-growth factor cross-talk during astroglial cell proliferation and differentiation in culture.

Isolation and Flow Cytometry Analysis of Intestinal Macrophages.

The intestinal macrophage pool represents the largest population of macrophages present within the body. Nevertheless, flow cytometry analysis of intestinal macrophages remains challenging due to historical lack of consensus on surface markers, variations in sample preparation, and a certain capriciousness of the isolation procedure itself. Furthermore, recent studies have uncovered a hitherto unknown heterogeneity of intestinal macrophages, accompanied by a vast increase of subset-identifying surface markers. Here, the isolation procedure for intestinal tissue for flow cytometry analysis is laid out, with particular attention toward the procedures for isolated intestinal layers, and a trouble-shooting section with strategies to avoid common pitfalls and mistakes.