Development of temperature-controlled batch and 3-column counter-current protein A system for improved therapeutic purification.

Maximizing product quality attributes by optimizing process parameters and performance attributes is a crucial aspect of bioprocess chromatography process design. Process parameters include but are not limited to bed height, eluate cut points, and elution pH. An under-characterized chromatography process parameter for protein A chromatography is process temperature. Here, we present a mechanistic understanding of the effects of temperature on the protein A purification of a monoclonal antibody (mAb) using a commercial chromatography resin for batch and continuous counter-current systems. A self-designed 3D-printed heating jacket controlled the 1 mL chromatography process temperature during the loading, wash, elution, and cleaning-in-place (CIP) steps. Batch loading experiments at 10, 20, and 30 °C demonstrated increased dynamic binding capacity (DBC) with temperature. The experimental data were fit to mechanistic and correlation-based models that predicted the optimal operating conditions over a range of temperatures. These model-based predictions optimized the development of a 3-column temperature-controlled periodic counter-current chromatography (TCPCC) and were validated experimentally. Operating a 3-column TCPCC at 30 °C led to a 47% increase in DBC relative to 20 °C batch chromatography. The DBC increase resulted in a two-fold increase in productivity relative to 20 °C batch. Increasing the number of columns to the TCPCC to optimize for increasing feed concentration resulted in further improvements to productivity. The feed-optimized TCPCC showed a respective two, three, and four-fold increase in productivity at feed concentrations of 1, 5, and 15 mg/mL mAb, respectively. The derived and experimentally validated temperature-dependent models offer a valuable tool for optimizing both batch and continuous chromatography systems under various operating conditions.

Clinical and Financial Impact of Rapid Antimicrobial Susceptibility Testing in Blood Cultures.

The rapid identification of pathogens that cause bloodstream infections plays a vital role in the modern clinical microbiology laboratory. Despite demonstrating a significant reduction in turnaround time and a significant effect on clinical decisions, most methods do not provide complete antimicrobial susceptibility testing (AST) information. We employed rapid identification (ID) and AST using the Accelerate PhenoTest on positive blood cultures containing Gram-negative bacilli. The length of stay (LOS) significantly decreased from an average of 12.1 days prior to implementation to 6.6 days post-implementation (p = 0.02), representing potential total savings of USD 666,208.00. All-cause mortality did not differ significantly, 27 (19%) versus 18 (12%), p = 0.11. We also observed an associated decrease in the use of broad-spectrum antimicrobials, including meropenem and quinolones. The implementation of a rapid ID and AST method, along with a well-established antimicrobial stewardship program, has the potential to decrease LOS, broad-spectrum antibiotic use, and costs to the healthcare system, with no observable impact on mortality.

Model free analysis of experimental residual dipolar couplings in small organic compounds.

Residual dipolar couplings (RDCs) contain information on the relative arrangement and dynamics of internuclear spin vectors in chemical compounds. Classically, RDC data is analyzed by fitting to structure models, while model-free approaches (MFA) directly relate RDCs to the corresponding internuclear vectors. The recently introduced software TITANIA implements the MFA and extracts structure and dynamics parameters directly from RDCs to facilitate de novo structure refinement for small organic compounds. Encouraged by our previous results on simulated data, we herein focus on the prerequisites and challenges faced when using purely experimental data for this approach. These concern mainly the fact that not all couplings are accessible in all media, leading to voids in the RDC matrix and the concomitant effects on the structure refinement. It is shown that RDC data sets obtained experimentally from currently available alignment media and measurement methods are of sufficient quality to allow relative configuration determination even when the relative configuration of the analyte is completely unknown.

TITANIA: Model-Free Interpretation of Residual Dipolar Couplings in the Context of Organic Compounds.

Residual dipolar couplings (RDCs) become increasingly important as additional NMR parameters in the structure elucidation of organic compounds but are usually used in fitting procedures to discriminate between (computed) structures that are in accordance with RDCs and others that can be ruled out. Thus, the determination of configurations requires prior structural information. The direct use of RDCs as restraints to construct structures based on RDCs has only recently begun also in organic compounds. No protocol has been published though that uses the vector and dynamics information available in multialignment data sets directly for the joint determination of conformation and configuration of organic compounds. This is proposed in the current study. We show that by employing these data, even a flat or random start structure converges into the correctly configured structure when employing multiple alignment data sets in our iterative procedure. The requirements in terms of the number of RDCs and alignment media necessary are discussed in detail.

Justice Sensitivity in Middle Childhood: Measurement and Location in the Temperamental and Social Skills Space.

Research suggested that justice sensitivity (JS)-the tendency to perceive and negatively respond to injustice-may already manifest in middle childhood, but empirical evidence is sparse. We, therefore, examined the measurement of JS in this age range and its associations with prosocial behavior, aggressive behavior, temperamental traits, and social skills. We had 361 children between 6 and 10 years of age and/or their parents rate the children's JS and its potential correlates. We replicated the JS-factor structure with three correlated subscales in both child and parent-ratings that showed strict measurement invariance. In line with previous findings in older age groups, victim JS positively predicted aggressive and negatively predicted prosocial behavior, whereas observer and perpetrator JS positively predicted prosocial and perpetrator JS negatively predicted aggressive behavior. The JS perspectives showed expected links with temperamental traits. All three subscales were positively related to empathy and theory of mind, but victim JS was negatively related to affective self-regulation. Findings suggest that interpersonal differences in JS may reliably and validly be measured in middle childhood and that JS is associated with aggressive and prosocial behavior already in childhood. Thus, future research should consider the role of JS for moral and personality development and developmental psychopathology.

Do employer-sponsored health insurance schemes affect the utilisation of medically trained providers and out-of-pocket payments among ready-made garment workers? A case-control study in Bangladesh.

OBJECTIVE: We estimated the effect of an employer-sponsored health insurance (ESHI) scheme on healthcare utilisation of medically trained providers and reduction of out-of-pocket (OOP) expenditure among ready-made garment (RMG) workers. DESIGN: We used a case-control study design with cross-sectional preintervention and postintervention surveys. SETTINGS: The study was conducted among workers of seven purposively selected RMG factories in Shafipur, Gazipur in Bangladesh. PARTICIPANTS: In total, 1924 RMG workers (480 from the insured and 482 from the uninsured, in each period) were surveyed from insured and uninsured RMG factories, respectively, in the preintervention (October 2013) and postintervention (April 2015) period. INTERVENTIONS: We tested the effect of a pilot ESHI scheme which was implemented for 1 year. OUTCOME MEASURES: The outcome measures were utilisation of medically trained providers and reduction of OOP expenditure among RMG workers. We estimated difference-in-difference (DiD) and applied two-part regression model to measure the association between healthcare utilisation, OOP payments and ESHI scheme membership while controlling for the socioeconomic characteristics of workers. RESULTS: The ESHI scheme increased healthcare utilisation of medically trained providers by 26.1% (DiD=26.1; p<0.01) among insured workers compared with uninsured workers. While accounting for covariates, the effect on utilisation significantly reduced to 18.4% (p<0.05). The DiD estimate showed that OOP expenditure among insured workers decreased by -3700 Bangladeshi taka and -1100 Bangladeshi taka compared with uninsured workers when using healthcare services from medically trained providers or all provider respectively, although not significant. The multiple two-part models also reported similar results. CONCLUSION: The ESHI scheme significantly increased utilisation of medically trained providers among RMG workers. However, it has no significant effect on OOP expenditure. It can be recommended that an educational intervention be provided to RMG workers to improve their healthcare-seeking behaviours and increase their utilisation of ESHI-designated healthcare providers while keeping OOP payments low.

Risk factors of brucellosis seropositivity in Bactrian camels of Mongolia.

BACKGROUND: More information on brucellosis epidemiology in Bactrian camels is needed due to their growing economic and livelihood importance for herders and renewed efforts in Mongolia to eliminate brucellosis through mass vaccination of ruminants excluding camels. Brucellosis prevalence in camels increased over the past two decades. Random multi-stage cluster surveys were done in the Eastern provinces of Dornod and Sukhbaatar in 2013 and 2014 and in the Southern & Western provinces of Dornogobi, Umnogobi and Khovd in 2014 and 2015. A total of 1822 camels, 1155 cattle, and 3023 small ruminant sera were collected and tested with the Rose Bengal Test. In addition, 195 vaginal swabs and 250 milk samples for bacteriological culture were taken from livestock with history of abortion. RESULTS: The overall apparent seroprevalence in camels was 2.3% (95% confidence interval 1.6-3.3). The main risk factor for camel seropositivity was being in an Eastern province when compared to Southern & Western provinces (odds ratio 13.2, 95% CI 5.3-32.4). Camel seroprevalences were stable over the two consecutive survey years, despite introduction of ruminant vaccination: 5.7% (95% CI 3.1-10.2%) and 5.8% (3.3-10.1%) in Eastern provinces and 0.4% (0.2-1.2%) and 0.5% (0.1-2.0%) in Southern & Western provinces. We isolated Brucella abortus from camels and cattle. Camel seropositivity was associated to keeping cattle together with camels. Monitoring of vaccination campaigns showed that coverage in cattle was insufficient because animals could not be adequately restrained. CONCLUSIONS: The present study reveals that brucellosis is present with important seroprevalence in Mongolian camels and was endemic in Eastern provinces. Camel herd seropositivity was most closely associated to infection in cattle. Longer term monitoring is needed to assess whether camel seroprevalance decreases with ongoing vaccination in Mongolia. This should be coupled with further confirmation on Brucella spp. isolates. To date, only Brucella abortus was isolated, but camels are also susceptible to Brucella melitensis. Clear verbal and written information on disease prevention in livestock and household members is important, particularly for remote camel herders who had only moderate knowledge on brucellosis epidemiology and preventive measures.

Influence of Luting Materials on the Retention of Cemented Implant-Supported Crowns: An In Vitro Study.

The retention force of cemented crowns on implant abutments with various luting materials was evaluated. Cobalt⁻chromium crowns were cemented onto tapered titanium abutments (Camlog) with eugenol-free temporary cement (RelyX TempBond NE), composite-based temporary cement (Bifix Temp), zinc phosphate cement (Harvard Cement), glass-ionomer cements (Meron, Fuji I), and resin-modified glass-ionomer cements (Fuji II, Fuji Plus, Ketac Cem Plus). Specimen aging via hydrostress was performed in artificial saliva at 37 °C for 14 days (S1), followed by hydrothermal stress with thermocycling (S2). The crowns were removed, and the force was recorded (T1). Subsequently, the crowns were recemented, aged, and removed, and the force was recorded (T2, T3). The retention forces differences were statistically significant according to the storage conditions at T1 (p = 0.002) and T3 (p = 0.0002). After aging (S1), Ketac Cem Plus had the highest retention force median value difference (T3 versus T1) (-773 N), whereas RelyX TempBond NE had the lowest (-146 N). After aging (S2), Meron had the highest retention force median value difference (-783 N), whereas RelyX TempBond NE had the lowest (-168 N). Recementation decreased the retention force of the implant-supported cobalt⁻chromium crowns cemented and recemented with the same luting materials. Luting materials (at T1) and aging conditions significantly impacted the retention force.

The Impact of Lens Opacity on SD-OCT Retinal Nerve Fiber Layer and Bruch's Membrane Opening Measurements Using the Anatomical Positioning System (APS).

Purpose: To evaluate the impact of lens opacity on retinal nerve fiber layer thickness (RNFLT) and Bruch's membrane opening (BMO) measurements. Methods: Fifty-nine randomly selected patients without any other relevant ocular pathology undergoing elective routine cataract surgery in two specialized eye clinics were enrolled. RNFLT, BMO area, and BMO minimum rim width (BMO-MRW) were assessed with the Heidelberg Engineering Spectralis OCT using the anatomical positioning system (APS) prior to and 1 day after cataract surgery using a ring scan at different eccentricities of the disc (3.5, 4.1 and 4.7 mm). Lens opacity was quantified using densitometry based on Scheimpflug images (Oculus Pentacam AXL). Results: RNFLT, BMO area, and BMO-MRW were virtually identical before and following removal of the cataractous lens. This held when assessed overall, within the six sectors for the 3.5-mm scan, or at any other eccentricity. Baseline RNFLT was not associated with lens opacity. Conclusions: Using the APS, RNFLT remained unchanged following cataract surgery, contrary to results reported by previous studies. Our results imply that the APS may have contributed to more precise spectral-domain optical coherence measurements, minimizing the influence of cataract on RNFLT and BMO assessments in our cohort.

Utility of a single nasal polymerase chain reaction assay in predicting absence of skin and environmental contamination in hospitalized patients with past methicillin-resistant Staphylococcus aureus.

We evaluated hospitalized patients with a history of methicillin-resistant Staphylococcus aureus (MRSA) for persistent colonization and need for contact precautions. Up to 3 daily cultures of nares, skin, and any present wounds were compared with a single nasal polymerase chain reaction (PCR) assay. Most patients (76.2%) were no longer colonized with MRSA. A single PCR assay was sufficient to exclude persistent colonization and environmental contamination and remove the contact precautions.

"Bringing down the flag": removing the contact precautions label for non-hospitalized patients with prior methicillin-resistant Staphylococcus aureus infection or colonization.

A policy consensus has not been reached regarding discontinuing contact precautions in patients with a history of methicillin-resistant Staphylococcus aureus (MRSA). We found that as many as 72% of outpatients flagged for past MRSA were no longer carriers, and a single nasal PCR test provided a reasonable negative predictive value for removing contact precautions.

Seroprevalence survey of brucellosis among rural people in Mongolia.

BACKGROUND: After the transition from socialism to a market economy in 1990, human brucellosis re-emerged in Mongolia. The aim of our study was to estimate a representative seroprevalence of Brucella spp. and to determine risk factors for brucellosis seropositivity among rural people. METHODS: A cross-sectional study with multistage random selection was conducted in eight provinces of Mongolia. Study participants were interviewed using a questionnaire to obtain their brucellosis history, current symptoms and likely risk factors. Blood samples were drawn to determine brucellosis seroprevalence. RESULTS: A total of 2856 randomly selected rural people aged four to 90 years were enrolled in the study. The seroprevalence of Brucella spp. was 11.1% (95% confidence interval [CI]: 10.0-12.1), ranging between 2.3% and 22.6% in the eight provinces; 39.2% (n = 609) of nomadic camps had at least one seropositive participant. Risk factors associated with brucellosis seropositivity were being older than 45 years (adjusted odds ratio [AOR] = 6.9, 95% CI = 5.1-8.7) and being a veterinarian (AOR = 2.8, 95% CI = 1.5-5.0). CONCLUSION: Our study confirms that human brucellosis seroprevalence among rural people in Mongolia is high. Human brucellosis can be effectively controlled if high-coverage livestock mass vaccination is implemented with a coverage survey after the vaccinations to ensure completeness. This mass vaccination should be accompanied by public awareness and educational programmes.

Restoration of the normal Clara cell phenotype after chronic allergic inflammation.

Bronchiolar Clara cells play a critical role in lung homoeostasis. The main goal of this study was to evaluate the effects of chronic allergy on these cells and the efficacy of budesonide (BUD) and montelukast (MK) in restoring their typical phenotypes after ovalbumin-induced chronic allergy in mice. Chronic allergy induced extensive bronchiolar Alcian blue-periodic acid-Schiff (AB/PAS)-positive metaplasia. In addition, cells accumulated numerous big electron-lucent granules negative for Clara cell main secretory protein (CC16), and consequently, CC16 was significantly reduced in bronchoalveolar lavage. A concomitant reduction in SP-D and CYP2E1 content was observed. The phenotypic changes induced by allergy were pharmacologically reversed by both treatments; MK was more efficient than BUD in doing so. MK decreased AB/PAS reactivity to control levels whereas they remained persistently elevated after BUD. Moreover, most non-ciliated cells recovered their normal morphology after MK, whereas for BUD normal cells coexisted with 'transitional' cells that contained remnant mucous granules and stained strongly for CC16 and SP-D. Glucocorticoids were also less able to reduce inflammatory infiltration and maintained higher percentage of neutrophils, which may have contributed to prolonged mucin expression. These results show that chronic allergy-induced mucous metaplasia of Clara cells affects their defensive mechanisms. However, anti-inflammatory treatments were able to re-establish the normal phenotype of Clara cell, with MK being more efficient at restoring a normal profile than BUD. This study highlights the role of epithelial cells in lung injuries and their contribution to anti-inflammatory therapies.

Cost estimate of bovine tuberculosis to Ethiopia.

While bovine tuberculosis (BTB) has been eliminated in some industrialized countries, it prevails worldwide, particularly in Africa. In Ethiopia, BTB is prevalent as numerous studies have shown its occurrence in livestock and in abattoirs but it has not been demonstrated in wildlife and only very few cases have been found in humans. The objective of this study is to estimate the cost of BTB to Ethiopia with the aim of informing Ethiopian policy on options for BTB control. BTB in livestock affects both animal productivity and herd demographic composition. The Livestock Development Planning System (LDPS2, FAO) was modified to allow for stochastic simulation of parameters. We performed an incremental cost of disease analysis, comparing livestock production with and without BTB. For the rural scenario we considered an endemically stable 4 % comparative intradermal test (CIDT) prevalence and for the urban scenario an endemically stable 32 % CIDT prevalence among cattle. The net present value of rural Ethiopian livestock products in 2005 is estimated at 65.7 billion (thousand million) Ethiopian Birr (95 % Confidence Interval (CI) 53.8-77.7 billion Birr), which is the equivalent of 7.5 billion US$ (95 %CI 6.1-8.9 billion US$) at a rate of 8.7 Birr per US$ in 2005. The cost of BTB ranges from 646 million Birr (75.2 million US$) in 2005 to 3.1 Billion Birr in 2011 (358 million US$) but is within the range of uncertainty of our estimate and can thus not be distinguished from zero. The cost of disease in the urban livestock production ranges from 5 to 42 million Birr (500,000-4.9 million US$) between 2005 and 2011 but is also within the range of uncertainty of our estimate. Our study shows no measurable loss in asset value or cost of disease due to BTB in rural and urban production systems in Ethiopia. This does not mean that there is not a real cost of disease, but the variability of the productivity parameters and prices are high and would require more precise estimates. This study does not preclude in any way the urgent need to control BTB in the urban dairy herd of Addis Ababa for other than financial reasons.

A "One Health" surveillance and control of brucellosis in developing countries: moving away from improvisation.

Although a "One Health" approach has been successfully implemented for emerging infectious zoonotic diseases with pandemic potential, we still lack a conceptual framework to address enzootic diseases like brucellosis. The vast majority of published brucellosis studies in the developing world rely solely on serology. An important shortcoming of brucellosis serology is the impossibility to infer which (smooth) Brucella spp. induced antibodies in the host. In this respect, mixed farming and especially raising small ruminants along with cattle, a common practice in the developing world, is reported to be a risk factor and a central question that has to be answered is whether cattle are infected with B. melitensis or with B. abortus or with both Brucella species. Therefore the isolation, identification and molecular characterization of Brucella spp. in human and the different livestock species needs to be undertaken to define a sound conceptual framework, identify the source of infection and plan appropriate control measures.

Aptamer-mediated blockade of IL4Rα triggers apoptosis of MDSCs and limits tumor progression.

In addition to promoting tumor progression and metastasis by enhancing angiogenesis and invasion, myeloid-derived suppressor cells (MDSC) and tumor-associated macrophage (TAM) also inhibit antitumor T-cell functions and limit the efficacy of immunotherapeutic interventions. Despite the importance of these leukocyte populations, a simple method for their specific depletion has not been developed. In this study, we generated an RNA aptamer that blocks the murine or human IL-4 receptor-α (IL4Rα or CD124) that is critical for MDSC suppression function. In tumor-bearing mice, this anti-IL4Rα aptamer preferentially targeted MDSCs and TAM and unexpectedly promoted their elimination, an effect that was associated with an increased number of tumor-infiltrating T cells and a reduction in tumor growth. Mechanistic investigations of aptamer-triggered apoptosis in MDSCs confirmed the importance of IL4Ra-STAT6 pathway activation in MDSC survival. Our findings define a straightforward strategy to deplete MDSCs and TAMs in vivo, and they strengthen the concept that IL4Rα signaling is pivotal for MDSC survival. More broadly, these findings suggest therapeutic strategies based on IL4Rα signaling blockades to arrest an important cellular mechanism of tumoral immune escape mediated by MDSCs and TAM in cancer.

Albuterol modulates its own transepithelial flux via changes in paracellular permeability.

Although inhaled bronchodilators are commonly used in the treatment of airway disease to dilate airway smooth muscle, little is known regarding the mechanisms that regulate albuterol movement across the epithelium to reach its target, the airway smooth muscle. Because the rate of onset depends on the transepithelial transport of albuterol, to determine the mechanisms that regulate the transepithelial movement of albuterol is essential. Human bronchial epithelial cells, fully redifferentiated in culture at the air-liquid interface, were used to study the cellular uptake and total transepithelial flux of (3)H-albuterol from the apical to the basolateral surfaces. (3)H-mannitol and transepithelial electrical resistance were used to quantify changes in paracellular permeability. The majority of albuterol flux across the epithelium occurred via the paracellular route. The cellular uptake of albuterol was found to be saturable, whereas transepithelial flux was not. Cellular uptake could be inhibited by the amino acids lysine and histidine, with no effect on net transepithelial flux. Transepithelial flux was altered by maneuvers that collapsed or disrupted intercellular junctions. Acidification, usually seen in exacerbations of airway disease, decreased albuterol flux. In addition, albuterol increased its own paracellular permeability. The ability of albuterol to modulate paracellular permeability was blocked by the ß(2)-adrenergic receptor-selective antagonist ICI 118551. Albuterol mainly crosses the epithelium via the paracellular pathway, but has the ability to modulate its own permeability through changes in the leakiness of tight junctions, which is modulated through the signaling of the ß(2)-adrenergic receptor.

Peptide-conjugated PAMAM dendrimer as a universal DNA vaccine platform to target antigen-presenting cells.

DNA-based vaccines hold promise to outperform conventional antigen-based vaccines by virtue of many unique features. However, DNA vaccines have thus far fallen short of expectations, due in part to poor targeting of professional antigen-presenting cells (APC) and low immunogenicity. In this study, we describe a new platform for effective and selective delivery of DNA to APCs in vivo that offers intrinsic immune-enhancing characteristics. This platform is based on conjugation of fifth generation polyamidoamine (G5-PAMAM) dendrimers, a DNA-loading surface, with MHC class II-targeting peptides that can selectively deliver these dendrimers to APCs under conditions that enhance their immune stimulatory potency. DNA conjugated with this platform efficiently transfected murine and human APCs in vitro. Subcutaneous administration of DNA-peptide-dendrimer complexes in vivo preferentially transfected dendritic cells (DC) in the draining lymph nodes, promoted generation of high affinity T cells, and elicited rejection of established tumors. Taken together, our findings show how PAMAM dendrimer complexes can be used for high transfection efficiency and effective targeting of APCs in vivo, conferring properties essential to generate effective DNA vaccines.

Acute inflammation promotes early cellular stimulation of the epithelial and stromal compartments of the rat prostate.

BACKGROUND: It has been proposed that prostatic inflammation plays a pivotal role in the pathophysiology of benign hyperplasia and prostate cancer. However, little information is available about the prostatic reaction to bacterial compounds in vivo. Our aim was therefore to evaluate the early effects of bacterial infection on rat ventral prostate compartments. METHODS: Using a rat model of acute bacterial prostatitis by Escherichia coli, we analyzed the histological and ultrastructural changes in the prostate at 24, 48, and 72 hr postinfection. Prostatic tissues were immunostained for prostatic binding protein (PBP), ACTA2, ErbB1, and ErbB2 receptors, TUNEL, and markers of cell proliferation. Dot and Western blots for PBP, ACTA2, ErbB1, ErbB2, and TGFbeta1 were also performed. RESULTS: The prostatic epithelium became hypertrophied, with increases in PBP and ErbB1 expression at 24 hr postinfection. Moreover, inflammation induced the expression of ErbB2, a receptor strongly involved in carcinogenesis. These alterations were more pronounced at 48 hr, but the epithelium also showed apoptosis and finally atrophy at 72 hr postinfection, with a decrease in PBP and ErbB receptors. Interestingly, the epithelial cells exhibited a high level of proliferation in response to the bacteria. The stromal reaction to acute inflammation was initially characterized by smooth muscle hypertrophy. Afterwards, muscle cells acquired a secretory phenotype, with a reduction in ACTA2 at 72 hr postinfection. CONCLUSIONS: Prostatic inflammation, even at the early stages, promotes atrophic and proliferative changes, and the upregulation of ErbB receptors together with dedifferentiation of smooth muscle cells. These data suggest that repetitive reinfections could lead to uncontrolled growth in the prostate gland.

In vivo visualization of dendritic cells, macrophages, and microglial cells responding to laser-induced damage in the fundus of the eye.

PURPOSE: To study the in vivo response of mononuclear phagocytes (i.e., dendritic cells [DCs] and macrophages [MPhis]) in the posterior eye segment after laser-induced injury, and to gain a better understanding of the role of these cells in inflammatory eye disease. METHODS: CX(3)CR1(GFP/+) knockin mice were used, in which DCs, MPhis, and microglia cells (microGCs) are constitutively fluorescent. These reporter mice were examined by a confocal scanning laser ophthalmoscope (cSLO) after argon laser coagulation. cSLO was complemented by fluorescence microscopy of retinal flatmounts and eye cryosections, to study cell morphology and location, and by multicolor flow cytometry, to determine the number and identity of the fluorescent cells. RESULTS: The retina of healthy reporter mice featured abundant fluorescent microGCs. After laser injury to the fundus, these cells accumulated and migrated laterally toward injury after 60 minutes. Distinctly shaped fluorescent cells accumulated within laser spots and were identified by flow cytometry and immunofluorescence microscopy as DCs and MPhis in the retina and choroid. The DCs rapidly disappeared from the retina, whereas the MPhis stayed longer. Choroidal infiltrates were detectable even 35 days after laser injury, in particular in larger spots resulting from higher laser intensity. In addition, nonfluorescent granulocytes were detected in the choroid. CONCLUSIONS: The synergistic use of ophthalmoscopy, flow cytometry, and immunofluorescence microscopy allows detailed dissection of the in vivo response of mononuclear phagocytes to laser injury of the fundus. The number of microGCs increased in the retina. DCs and MPhis were present in the retina and choroid infiltrate. MPhis and granulocytes persisted in the choroid infiltrate longer than previously thought.