Vaccine-Associated Maintenance of Epithelial Integrity Correlated With Protection Against Virus Entry.

To identify the mechanisms by which human immunodeficiency virus type 1 (HIV-1) might penetrate the epithelial barrier during sexual transmission to women and the mechanisms of vaccine-associated protection against entry, we characterized early epithelial responses to vaginal inoculation of simian immunodeficiency virus strain mac251 (SIVmac251) in naive or SIVmac239Δnef-vaccinated rhesus macaques. Vaginal inoculation induced an early stress response in the cervicovaginal epithelium, which was associated with impaired epithelial integrity, damaged barrier function, and virus and bacterial translocation. In vaccinated animals, early stress responses were suppressed, and the maintenance of epithelial barrier integrity correlated with prevention of virus entry. These vaccine-protective effects were associated with a previously described mucosal system for locally producing and concentrating trimeric gp41 antibodies at the mucosal interface and with formation of SIV-specific immune complexes that block the stress responses via binding to the epithelial receptor FCGR2B and subsequent inhibitory signaling. Thus, blocking virus entry may be one protective mechanism by which locally concentrated non-neutralizing Ab might prevent HIV sexual transmission to women.

Strength and Viscosity Effects on Perturbed Shock Front Stability in Metals.

Computational modeling and experimental measurements on metal samples subject to a laser-driven, ablative Richtmyer-Meshkov instability showed differences between viscosity and strength effects. In particular, numerical and analytical solutions, coupled with measurements of fed-through perturbations, generated by perturbed shock fronts onto initially flat surfaces, show promise as a validation method for models of deviatoric response in the postshocked material. Analysis shows that measurements of shock perturbation amplitudes at low sample thickness-to-wavelength ratios are not enough to differentiate between strength and viscosity effects, but that surface displacement data of the fed-through perturbations appears to resolve the ambiguity. Additionally, analytical and numerical results show shock front perturbation evolution dependence on initial perturbation amplitude and wavelength is significantly different in viscous and materials with strength, suggesting simple experimental geometry changes should provide data supporting one model or the other.

Control of Escherichia coli O157 on beef at 37, 22 and 4 °C by T5-, T1-, T4-and O1-like bacteriophages.

Efficacy of four bacteriophages (phages) and a cocktail for biocontrol of Escherichia coli O157 was assessed on beef samples stored at 4, 22 and 37 °C. Samples (3 × 3 × 1 cm) were contaminated with E. coli O157 (10(4) CFU/cm(2)) and treated with single phages: T5-like (T5), T1-like (T1), T4-like (T4) and O1-like (O1), or a cocktail at two titers: multiplicity of infection (MOI) = 1000 and MOI = 10. In contrast to previous studies, use of virucidal solution prevented over-estimation of phage efficacy. Irrespective of temperature and MOIs, T5 was most (P < 0.001) and O1 least (P < 0.05) effective for biocontrol of E. coli O157, with relative efficacy of other phages temperature dependent. At 4 °C, T1 (P < 0.05) and cocktail (P < 0.001) were more effective than T4. In contrast, T4 was equally (P = 0.08, at 37 °C) or less effective (P = 0.003, at 22 °C) than T5. Phages were more effective (P < 0.001) against E. coli O157 at warmer temperatures and high MOI. As the beef supply chain includes hours of storage or transport at temperatures near 4 °C, this study demonstrates phages could significantly reduce E. coli O157 during this period.

Differing populations of endemic bacteriophages in cattle shedding high and low numbers of Escherichia coli O157:H7 bacteria in feces.

The objectives of this study were to identify endemic bacteriophages (phages) in the feedlot environment and determine relationships of these phages to Escherichia coli O157:H7 from cattle shedding high and low numbers of naturally occurring E. coli O157:H7. Angus crossbred steers were purchased from a southern Alberta (Canada) feedlot where cattle excreting ≥ 10(4) CFU · g(-1) of E. coli O157:H7 in feces at a single time point were identified as supershedders (SS; n = 6), and cattle excreting <10(4) CFU · g(-1) of feces were identified as low shedders (LS; n = 5). Fecal pats or fecal grabs were collected daily from individual cattle for 5 weeks. E. coli O157:H7 in feces was detected by immunomagnetic separation and enumerated by direct plating, and phages were isolated using short- and overnight-enrichment methods. The total prevalence of E. coli O157:H7 isolated from feces was 14.4% and did not differ between LS and SS (P = 0.972). The total prevalence of phages was higher in the LS group (20.9%) than in the SS group (8.3%; P = 0.01). Based on genome size estimated by pulsed-field gel electrophoresis and morphology determined by transmission electron microscopy, T4- and O1-like phages of Myoviridae and T1-like phage of Siphoviridae were isolated. Compared to T1- and O1-like phages, T4-like phages exhibited a broad host range and strong lytic capability when targeting E. coli O157:H7. Moreover, the T4-like phages were more frequently isolated from feces of LS than SS, suggesting that endemic phages may impact the shedding dynamics of E. coli O157:H7 in cattle.

Detection and prevalence of verotoxin-producing Escherichia coli O157 and non-O157 serotypes in a Canadian watershed.

Verotoxin-producing Escherichia coli (VTEC) strains are the cause of food-borne and waterborne illnesses around the world. Traditionally, surveillance of the human population as well as the environment has focused on the detection of E. coli O157:H7. Recently, increasing recognition of non-O157 VTEC strains as human pathogens and the German O104:H4 food-borne outbreak have illustrated the importance of considering the broader group of VTEC organisms from a public health perspective. This study presents the results of a comparison of three methods for the detection of VTEC in surface water, highlighting the efficacy of a direct VT immunoblotting method without broth enrichment for detection and isolation of O157 and non-O157 VTEC strains. The direct immunoblot method eliminates the need for an enrichment step or the use of immunomagnetic separation. This method was developed after 4 years of detecting low frequencies (1%) of E. coli O157:H7 in surface water in a Canadian watershed, situated within one of the FoodNet Canada integrated surveillance sites. By the direct immunoblot method, VTEC prevalence estimates ranged from 11 to 35% for this watershed, and E. coli O157:H7 prevalence increased to 4% (due to improved method sensitivity). This direct testing method provides an efficient means to enhance our understanding of the prevalence and types of VTEC in the environment. This study employed a rapid evidence assessment (REA) approach to frame the watershed findings with watershed E. coli O157:H7 prevalences reported in the literature since 1990 and the knowledge gap with respect to VTEC detection in surface waters.

Pressurized H2 RF cavities in ionizing beams and magnetic fields.

A major technological challenge in building a muon cooling channel is operating rf cavities in multitesla external magnetic fields. We report the first proof-of-principle experiment of a high pressure gas-filled rf cavity for use with intense ionizing beams and strong external magnetic fields. rf power consumption by beam-induced plasma is investigated with hydrogen and deuterium gases with pressures between 20 and 100 atm and peak rf gradients between 5 and 50 MV/m. The low pressure case agrees well with an analytical model based on electron and ion mobilities. Varying concentrations of oxygen gas are investigated to remove free electrons from the cavity and reduce the rf power consumption. Measurements of the electron attachment time to oxygen and rate of ion-ion recombination are also made. Additionally, we demonstrate the operation of the gas-filled rf cavity in a solenoidal field of up to 3 T, finding no major magnetic field dependence. All these results indicate that a high pressure gas-filled cavity is a viable technology for muon ionization cooling.

Bright laser-driven neutron source based on the relativistic transparency of solids.

Neutrons are unique particles to probe samples in many fields of research ranging from biology to material sciences to engineering and security applications. Access to bright, pulsed sources is currently limited to large accelerator facilities and there has been a growing need for compact sources over the recent years. Short pulse laser driven neutron sources could be a compact and relatively cheap way to produce neutrons with energies in excess of 10 MeV. For more than a decade experiments have tried to obtain neutron numbers sufficient for applications. Our recent experiments demonstrated an ion acceleration mechanism based on the concept of relativistic transparency. Using this new mechanism, we produced an intense beam of high energy (up to 170 MeV) deuterons directed into a Be converter to produce a forward peaked neutron flux with a record yield, on the order of 10(10) n/sr. We present results comparing the two acceleration mechanisms and the first short pulse laser generated neutron radiograph.

Gene cluster conferring streptomycin, sulfonamide, and tetracycline resistance in Escherichia coli O157:H7 phage types 23, 45, and 67.

Multidrug resistance to streptomycin, sulfonamide, and tetracycline (AMR-SSuT) was identified in 156 of 171 isolates of Escherichia coli O157:H7 of phage types 23, 45, and 67. In 154 AMR-SSuT isolates, resistance was encoded by strA, strB, sul2, and tet(B), which in 59 of 63 tested isolates were found clustered together on the chromosome within the cdiA locus.

Assembly of focal adhesions: progress, paradigms, and portents.

Receptor-mediated assembly of an adhesion plaque occurs through an ordered series of steps, and intermediate assemblies can be identified. The recent demonstration of some of these partial reactions in permeabilized cells predicts that cell-free reconstitution of adhesion plaque assembly is an attainable goal. Newly discovered cryptic actin-binding sites in vinculin and ezrin, two proteins recruited to adhesion sites, suggest that actin-binding proteins are targets for the signals generated by adhesion receptors.

Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species.

We previously described a method for isolating murine hematopoietic stem cells capable of reconstituting lethally irradiated recipients, which depends solely on dual-wavelength flow cytometric analysis of murine bone marrow cells stained with the fluorescent DNA-binding dye Hoechst 33342. This method, which appears to rely on the differential ability of stem cells to efflux the Hoechst dye, defines an extremely small and homogeneous population of cells (termed SP cells). We show here that dual-wavelength analysis of Hoechst dye-stained human, rhesus and miniature swine bone marrow cells reveals a small, distinct population of cells that efflux the dye in a manner identical to murine SP cells. Like the murine SP cells, both human and rhesus SP cells are primarily CD34-negative and lineage marker-negative. In vitro culture studies demonstrated that rhesus SP cells are highly enriched for long-term culture-initiating cells (LTC-ICs), an indicator of primitive hematopoietic cells, and have the capacity for differentiation into T cells. Although rhesus SP cells do not initially possess any hematopoietic colony-forming capability, they acquire the ability to form colonies after long-term culture on bone marrow stroma, coincident with their conversion to a CD34-positive phenotype. These studies suggest the existence of a hitherto unrecognized population of hematopoietic stem cells that lack the CD34 surface marker classically associated with primitive hematopoietic cells.

Neutralizing antibody-independent containment of immunodeficiency virus challenges by DNA priming and recombinant pox virus booster immunizations.

Eight different protocols were compared for their ability to raise protection against immunodeficiency virus challenges in rhesus macaques. The most promising containment of challenge infections was achieved by intradermal DNA priming followed by recombinant fowl pox virus booster immunizations. This containment did not require neutralizing antibody and was active for a series of challenges ending with a highly virulent virus with a primary isolate envelope heterologous to the immunizing strain.

The accuracy of helium ion CT based particle therapy range prediction: an experimental study comparing different particle and x-ray CT modalities.

This work provides a quantitative assessment of helium ion CT (HeCT) for particle therapy treatment planning. For the first time, HeCT based range prediction accuracy in a heterogeneous tissue phantom is presented and compared to single-energy x-ray CT (SECT), dual-energy x-ray CT (DECT) and proton CT (pCT). HeCT and pCT scans were acquired using the US pCT collaboration prototype particle CT scanner at the Heidelberg Ion-Beam Therapy Center. SECT and DECT scans were done with a Siemens Somatom Definition Flash and converted to RSP. A Catphan CTP404 module was used to study the RSP accuracy of HeCT. A custom phantom of 20 cm diameter containing several tissue equivalent plastic cubes was used to assess the spatial resolution of HeCT and compare it to DECT. A clinically realistic heterogeneous tissue phantom was constructed using cranial slices from a pig head placed inside a cylindrical phantom (ø150 mm). A proton beam (84.67 mm range) depth-dose measurement was acquired using a stack of GafchromicTM EBT-XD films in a central dosimetry insert in the phantom. CT scans of the phantom were acquired with each modality, and proton depth-dose estimates were simulated based on the reconstructions. The RSP accuracy of HeCT for the plastic phantom was found to be 0.3 ± 0.1%. The spatial resolution for HeCT of the cube phantom was 5.9 ± 0.4 lp cm-1for central, and 7.6 ± 0.8 lp cm-1for peripheral cubes, comparable to DECT spatial resolution (7.7 ± 0.3 lp cm-1and 7.4 ± 0.2 lp cm-1, respectively). For the pig head, HeCT, SECT, DECT and pCT predicted range accuracy was 0.25%, -1.40%, -0.45% and 0.39%, respectively. In this study, HeCT acquired with a prototype system showed potential for particle therapy treatment planning, offering RSP accuracy, spatial resolution, and range prediction accuracy comparable to that achieved with a commercial DECT scanner. Still, technical improvements of HeCT are needed to enable clinical implementation.

Identification of overlapping HLA class I-restricted cytotoxic T cell epitopes in a conserved region of the human immunodeficiency virus type 1 envelope glycoprotein: definition of minimum epitopes and analysis of the effects of sequence variation.

Although the immunologic basis of protective immunity in human immunodeficiency virus type 1 (HIV-1) infection has not yet been defined, virus-specific cytotoxic T lymphocytes (CTL) are likely to be an important host defense and may be a critical feature of an effective vaccine. These observations, along with the inclusion of the HIV-1 envelope in the majority of vaccine candidates presently in clinical trials, underscore the importance of the precise characterization of the cellular immune responses to this protein. Although humoral immune responses to the envelope protein have been extensively characterized, relatively little information is available regarding the envelope epitopes recognized by virus-specific CTL and the effects of sequence variation within these epitopes. Here we report the identification of two overlapping CTL epitopes in a highly conserved region of the HIV-1 transmembrane envelope protein, gp41, using CTL clones derived from two seropositive subjects. An eight-amino acid peptide was defined as the minimum epitope recognized by HLA-B8-restricted CTL derived from one subject, and in a second subject, an overlapping nine-amino acid peptide was identified as the minimal epitope for HLA-B14-restricted CTL clones. Selected single amino acid substitutions representing those found in naturally occurring HIV-1 isolates resulted in partial to complete loss of recognition of these epitopes. These data indicate the presence of a highly conserved region in the HIV-1 envelope glycoprotein that is immunogenic for CTL responses. In addition, they suggest that natural sequence variation may lead to escape from immune detection by HIV-1-specific CTL. Since the region containing these epitopes has been previously shown to contain an immunodominant B cell epitope and also overlaps with a major histocompatibility complex class II T cell epitope recognized by CD4+ CTL from HIV-1 rgp160 vaccine recipients, it may be particularly important for HIV-1 vaccine development. Finally, the identification of minimal CTL epitopes presented by class I HLA molecules should facilitate the definition of allele-specific motifs.

Naturally processed viral peptides recognized by cytotoxic T lymphocytes on cells chronically infected by human immunodeficiency virus type 1.

We have established long-term cultures of several cell lines stably and uniformly expressing human immunodeficiency virus type 1 (HIV-1) in order to (a) identify naturally processed HIV-1 peptides recognized by cytotoxic T lymphocytes (CTL) from HIV-1-seropositive individuals and (b) consider the hypothesis that naturally occurring epitope densities on HIV-infected cells may limit their lysis by CTL. Each of two A2-restricted CD8+ CTL specific for HIV-1 gag or reverse transcriptase (RT) recognized a single naturally processed HIV-1 peptide in trifluoroacetic acid (TFA) extracts of infected cells: gag 77-85 (SLYNTVATL) or RT 476-484 (ILKEPVHGV). Both processed peptides match the synthetic peptides that are optimally active in cytotoxicity assays and have the consensus motif described for A2-associated peptides. Their abundances were approximately 400 and approximately 12 molecules per infected Jurkat-A2 cell, respectively. Other synthetic HIV-1 peptides active at subnanomolar concentrations were not present in infected cells. Except for the antigen processing mutant line T2, HIV-infected HLA-A2+ cell lines were specifically lysed by both A2-restricted CTL, although infected Jurkat-A2 cells were lysed more poorly by RT-specific CTL than by gag-specific CTL, suggesting that low cell surface density of a natural peptide may limit the effectiveness of some HIV-specific CTL despite their vigorous activity against synthetic peptide-treated target cells.

Longitudinal analysis of T cell receptor (TCR) gene usage by human immunodeficiency virus 1 envelope-specific cytotoxic T lymphocyte clones reveals a limited TCR repertoire.

Human immunodeficiency virus 1 (HIV-1) infection is associated with a vigorous cellular immune response that allows detection of cytotoxic T lymphocyte (CTL) activity using freshly isolated peripheral blood mononuclear cells (PBMC). Although restricting class I antigens and epitopes recognized by HIV-1-specific CTL have been defined, the effector cells mediating this vigorous response have been characterized less well. Specifically, no studies have addressed the breadth and duration of response to a defined epitope. In the present study, a longitudinal analysis of T cell receptor (TCR) gene usage by CTL clones was performed in a seropositive person using TCR gene sequences as a means of tracking responses to a well-defined epitope in the glycoprotein 41 transmembrane protein. 10 CTL clones specific for this human histocompatibility leukocyte antigen-B14-restricted epitope were isolated at multiple time points over a 31-mo period. All clones were derived from a single asymptomatic HIV-1-infected individual with a vigorous response to this epitope that was detectable using unstimulated PBMC. Polymerase chain reaction amplification using V alpha and V beta family-specific primers was performed on each clone, followed by DNA sequencing of the V-D-J regions. All 10 clones utilized V alpha 14 and V beta 4 genes. Sequence analysis of the TCR revealed the first nine clones isolated to also be identical at the nucleotide level. The TCR-alpha junctional region sequence of the tenth clone was identical to the junctional region sequences of the other nine, but this clone utilized distinct D beta and J beta gene segments. This study provides evidence that the observed high degree of HIV-1-specific CTL activity may be due to monoclonal or oligoclonal expansion of specific effector cells, and that progeny of a particular CTL clone may persist for prolonged periods in vivo in the presence of a chronic productive viral infection. The observed limited TCR diversity against an immunodominant epitope may limit recognition of virus variants with mutations in regions interacting with the TCR, thereby facilitating immune escape.

T cell receptor usage and fine specificity of human immunodeficiency virus 1-specific cytotoxic T lymphocyte clones: analysis of quasispecies recognition reveals a dominant response directed against a minor in vivo variant.

Numerous virus-specific, class I-restricted cytotoxic T lymphocyte (CTL) epitopes have been identified, yet little information is available regarding the specificity of the CTL response in persons of the same human histocompatibility leukocyte antigen (HLA) type. In this study, the human immunodeficiency virus (HIV) 1 envelope-specific CTL response was evaluated in five HLA-B14-positive persons. CTL responses specific for a previously described nine-amino acid epitope in gp41 (aa 584-592, ERYLKDQQL) could be identified in all subjects, and CTL clones specific for this epitope could be isolated from four persons. Despite heterogeneous T cell receptor usage, the fine specificity of the clones was similar, as defined by recognition of alanine-substituted peptides as well as peptides representing natural HIV-1 sequence variants. Correlation with in vivo virus sequences revealed that the dominant species in two of the subjects represented poorly recognized variants, with a K-->Q substitution at amino acid 588, whereas no variants were observed in the other two subjects. Although clonal type-specific responses to these dominant variants could be identified, the magnitude of these responses remained small, and the dominant CTL response was directed at the minor in vivo variant. These studies indicate that despite similar epitope-specific immunologic pressure in persons of the same HLA type, the in vivo quasispecies may differ, and that the major in vivo immune response to a given CTL epitope can be directed at a minor variant.

Early regeneration of thymic progenitors in rhesus macaques infected with simian immunodeficiency virus.

The thymus plays a critical role in the maturation and production of T lymphocytes and is a target of infection by human immunodeficiency virus (HIV) and the related simian immunodeficiency virus (SIV). Using the SIV/macaque model of AIDS, we examined the early effects of SIV on the thymus. We found that thymic infection by SIV resulted in increased apoptosis 7-14 d after infection, followed by depletion of thymocyte progenitors by day 21. A marked rebound in thymocyte progenitors occurred by day 50 and was accompanied by increased levels of cell proliferation in the thymus. Our results demonstrate a marked increase in thymic progenitor activity very early in the course of SIV infection, long before marked declines in peripheral CD4(+) T cell counts.

Oral delivery systems for encapsulated bacteriophages targeted at Escherichia coli O157:H7 in feedlot cattle.

Bacteriophages are natural predators of bacteria and may mitigate Escherichia coli O157:H7 in cattle and their environment. As bacteriophages targeted to E. coli O157:H7 (phages) lose activity at low pH, protection from gastric acidity may enhance efficacy of orally administered phages. Polymer encapsulation of four phages, wV8, rV5, wV7, and wV11, and exposure to pH 3.0 for 20 min resulted in an average 13.6% recovery of phages after release from encapsulation at pH 7.2. In contrast, untreated phages under similar conditions had a complete loss of activity. Steers (n = 24) received 10(11) CFU of naladixic acid-resistant E. coli O157:H7 on day 0 and were housed in six pens of four steers. Two pens were control (naladixic acid-resistant E. coli O157:H7 only), and the remaining pens received polymer-encapsulated phages (Ephage) on days -1, 1, 3, 6, and 8. Two pens received Ephage orally in gelatin capsules (bolus; 10(10) PFU per steer per day), and the remaining two pens received Ephage top-dressed on their feed (feed; estimated 10(11) PFU per steer per day). Shedding of E. coli O157:H7 was monitored for 10 weeks by collecting fecal grab and hide swab samples. Acceptable activity of mixed phages at delivery to steers was found for bolus and feed, averaging 1.82 and 1.13 x 10(9) PFU/g, respectively. However, Ephage did not reduce shedding of naladixic acid-resistant E. coli O157:H7, although duration of shedding was reduced by 14 days (P < 0.1) in bolus-fed steers as compared with control steers. Two successful systems for delivery of Ephage were developed, but a better understanding of phage-E. coli O157:H7 ecology is required to make phage therapy a viable strategy for mitigation of this organism in feedlot cattle.

An optimization algorithm for dose reduction with fluence-modulated proton CT.

PURPOSE: Fluence-modulated proton computed tomography (FMpCT) using pencil beam scanning aims at achieving task-specific image noise distributions by modulating the imaging proton fluence spot-by-spot based on an object-specific noise model. In this work, we present a method for fluence field optimization and investigate its performance in dose reduction for various phantoms and image variance targets. METHODS: The proposed method uses Monte Carlo simulations of a proton CT (pCT) prototype scanner to estimate expected variance levels at uniform fluence. Using an iterative approach, we calculate a stack of target variance projections that are required to achieve the prescribed image variance, assuming a reconstruction using filtered backprojection. By fitting a pencil beam model to the ratio of uniform fluence variance and target variance, relative weights for each pencil beam can be calculated. The quality of the resulting fluence modulations is evaluated by scoring imaging doses and comparing them to those at uniform fluence, as well as evaluating conformity of the achieved variance with the prescription. For three different phantoms, we prescribed constant image variance as well as two regions-of-interest (ROI) imaging tasks with inhomogeneous image variance. The shape of the ROIs followed typical beam profiles for proton therapy. RESULTS: Prescription of constant image variance resulted in a dose reduction of 8.9% for a homogeneous water phantom compared to a uniform fluence scan at equal peak variance level. For a more heterogeneous head phantom, dose reduction increased to 16.0% for the same task. Prescribing two different ROIs resulted in dose reductions between 25.7% and 40.5% outside of the ROI at equal peak variance levels inside the ROI. Imaging doses inside the ROI were increased by 9.2% to 19.2% compared to the uniform fluence scan, but can be neglected assuming that the ROI agrees with the therapeutic dose region. Agreement of resulting variance maps with the prescriptions was satisfactory. CONCLUSIONS: We developed a method for fluence field optimization based on a noise model for a real scanner used in pCT. We demonstrated that it can achieve prescribed image variance targets. A uniform fluence field was shown not to be dose optimal and dose reductions achievable with the proposed method for FMpCT were considerable, opening an interesting perspective for image guidance and adaptive therapy.

Experimental realization of dynamic fluence field optimization for proton computed tomography.

Proton computed tomography (pCT) has high accuracy and dose efficiency in producing spatial maps of the relative stopping power (RSP) required for treatment planning in proton therapy. With fluence-modulated pCT (FMpCT), prescribed noise distributions can be achieved, which allows to decrease imaging dose by employing object-specific dynamically modulated fluence during the acquisition. For FMpCT acquisitions we divide the image into region-of-interest (ROI) and non-ROI volumes. In proton therapy, the ROI volume would encompass all treatment beams. An optimization algorithm then calculates dynamically modulated fluence that achieves low prescribed noise inside the ROI and high prescribed noise elsewhere. It also produces a planned noise distribution, which is the expected noise map for that fluence, as calculated with a Monte Carlo simulation. The optimized fluence can be achieved by acquiring pCT images with grids of intensity modulated pencil beams. In this work, we interfaced the control system of a clinical proton beam line to deliver the optimized fluence. Using three phantoms we acquired images with uniform fluence, with a constant noise prescription, and with an FMpCT task. Image noise distributions as well as fluence maps were compared to the corresponding planned distributions as well as to the prescription. Furthermore, we propose a correction method that removes image artifacts stemming from the acquisition with pencil beams having a spatially varying energy distribution that is not seen in clinical operation. RSP accuracy of FMpCT scans was compared to uniform scans and was found to be comparable to standard pCT scans. While we identified technical improvements for future experimental acquisitions, in particular related to an unexpected pencil beam size reduction and a misalignment of the fluence pattern, agreement with the planned noise was satisfactory and we conclude that FMpCT optimized for specific image noise prescriptions is experimentally feasible.