CXCL8/CXCR2 signaling mediates bone marrow fibrosis and is a therapeutic target in myelofibrosis.

Proinflammatory signaling is a hallmark feature of human cancer, including in myeloproliferative neoplasms (MPNs), most notably myelofibrosis (MF). Dysregulated inflammatory signaling contributes to fibrotic progression in MF; however, the individual cytokine mediators elicited by malignant MPN cells to promote collagen-producing fibrosis and disease evolution are yet to be fully elucidated. Previously, we identified a critical role for combined constitutive JAK/STAT and aberrant NF-κB proinflammatory signaling in MF development. Using single-cell transcriptional and cytokine-secretion studies of primary cells from patients with MF and the human MPLW515L (hMPLW515L) murine model of MF, we extend our previous work and delineate the role of CXCL8/CXCR2 signaling in MF pathogenesis and bone marrow fibrosis progression. Hematopoietic stem/progenitor cells from patients with MF are enriched for a CXCL8/CXCR2 gene signature and display enhanced proliferation and fitness in response to an exogenous CXCL8 ligand in vitro. Genetic deletion of Cxcr2 in the hMPLW515L-adoptive transfer model abrogates fibrosis and extends overall survival, and pharmacologic inhibition of the CXCR1/2 pathway improves hematologic parameters, attenuates bone marrow fibrosis, and synergizes with JAK inhibitor therapy. Our mechanistic insights provide a rationale for therapeutic targeting of the CXCL8/CXCR2 pathway among patients with MF.

Application limits of the scaling relations for Monte Carlo simulations in diffuse optics. Part 1: theory.

Monte Carlo (MC) is a powerful tool to study photon migration in scattering media, yet quite time-consuming to solve inverse problems. To speed up MC-simulations, scaling relations can be applied to an existing initial MC-simulation to generate a new data-set with different optical properties. We named this approach trajectory-based since it uses the knowledge of the detected photon trajectories of the initial MC-simulation, in opposition to the slower photon-based approach, where a novel MC-simulation is rerun with new optical properties. We investigated the convergence and applicability limits of the scaling relations, both related to the likelihood that the sample of trajectories considered is representative also for the new optical properties. For absorption, the scaling relation contains smoothly converging Lambert-Beer factors, whereas for scattering it is the product of two quickly diverging factors, whose ratio, for NIRS cases, can easily reach ten orders of magnitude. We investigated such instability by studying the probability-distribution for the number of scattering events in trajectories of given length. We propose a convergence test of the scattering scaling relation based on the minimum-maximum number of scattering events in recorded trajectories. We also studied the dependence of MC-simulations on optical properties, most critical in inverse problems, finding that scattering derivatives are ascribed to small deviations in the distribution of scattering events from a Poisson distribution. This paper, which can also serve as a tutorial, helps to understand the physics of the scaling relations with the causes of their limitations and devise new strategies to deal with them.

Two-layer reconstruction of Raman spectra in diffusive media based on an analytical model in the time domain.

We derive and validate an analytical model that describes the migration of Raman scattered photons in two-layer diffusive media, based on the diffusion equation in the time domain. The model is derived under a heuristic approximation that background optical properties are identical on the excitation and Raman emission wavelengths. Methods for the reconstruction of two-layer Raman spectra have been developed, tested in computer simulations and validated on tissue-mimicking phantom measurements data. Effects of different parameters were studied in simulations, showing that the thickness of the top layer and number of detected photon counts have the most significant impact on the reconstruction. The concept of quantitative, mathematically rigorous reconstruction using the proposed model was finally proven on experimental measurements, by successfully separating the spectra of silicone and calcium carbonate (calcite) layers, showing the potential for further development and eventual application in clinical diagnostics.

Patients with hypercortisolemic Cushing disease possess a distinct class of hematopoietic progenitor cells leading to erythrocytosis.

Although human cell cultures stimulated with dexamethasone suggest that the glucocorticoid receptor (GR) activates stress erythropoiesis, the effects of GR activation on erythropoiesis in vivo remain poorly understood. We characterized the phenotype of a large cohort of patients with Cushing disease, a rare condition associated with elevated cortisol levels. Results from hypercortisolemic patients with active Cushing disease were compared with those obtained from eucortisolemic patients after remission and from volunteers without the disease. Patients with active Cushing disease exhibited erythrocytosis associated with normal hemoglobin F levels. In addition, their blood contained elevated numbers of GR-induced CD163+ monocytes and a unique class of CD34+ cells expressing CD110, CD36, CD133 and the GR-target gene CXCR4. When cultured, these CD34+ cells generated similarly large numbers of immature erythroid cells in the presence and absence of dexamethasone, with raised expression of the GR-target gene GILZ. Of interest, blood from patients with Cushing disease in remission maintained high numbers of CD163+ monocytes and, although their CD34+ cells had a normal phenotype, these cells were unresponsive to added dexamethasone. Collectively, these results indicate that chronic exposure to excess glucocorticoids in vivo leads to erythrocytosis by generating erythroid progenitor cells with a constitutively active GR. Although remission rescues the erythrocytosis and the phenotype of the circulating CD34+ cells, a memory of other prior changes is maintained in remission.

Monte Carlo simulations in anomalous radiative transfer: tutorial.

Anomalous radiative transfer (ART) theory represents a generalization of classical radiative transfer theory. The present tutorial aims to show how Monte Carlo (MC) codes describing the transport of photons in anomalous media can be implemented. We show that the heart of the method involves suitably describing, in a "non-classical" manner, photon steps starting from fixed light sources or from boundaries separating regions of the medium with different optical properties. To give a better sense of the importance of these particular photon step lengths, we also show numerically that the described approach is essential in preserving the invariance property for light propagation. An interesting byproduct of the MC method for ART is that it allows us to simplify the structure of "classical" MC codes, utilized, for example, in biomedical optics.

Direct Measurement of the Reduced Scattering Coefficient by a Calibrated Random Laser Sensor.

The research in optical sensors has been largely encouraged by the demand for low-cost and less or non-invasive new detection strategies. The invention of the random laser has opened a new frontier in optics, providing also the opportunity to explore new possibilities in the field of sensing, besides several different and peculiar phenomena. The main advantage in exploiting the physical principle of the random laser in optical sensors is due to the presence of the stimulated emission mechanism, which allows amplification and spectral modification of the signal. Here, we present a step forward in the exploitation of this optical phenomenon by a revisitation of a previous experimental setup, as well as the measurement method, in particular to mitigate the instability of the results due to shot-to-shot pump energy fluctuations. In particular, the main novelties of the setup are the use of optical fibers, a reference sensor, and a peristaltic pump. These improvements are devoted to: eliminating optical beam alignment issues; improving portability; mitigating the variation in pump energy and gain medium performances over time; realizing an easy and rapid change of the sensed medium. The results showed that such a setup can be considered a prototype for a portable device for directly measuring the scattering of liquid samples, without resorting to complicated numerical or analytic inversion procedures of the measured data, once the suitable calibration of the system is performed.

Novel targets to cure primary myelofibrosis from studies on Gata1low mice.

In 2002, we discovered that mice carrying the hypomorphic Gata1low mutation that reduces expression of the transcription factor GATA1 in megakaryocytes (Gata1low mice) develop myelofibrosis, a phenotype that recapitulates the features of primary myelofibrosis (PMF), the most severe of the Philadelphia-negative myeloproliferative neoplasms (MPNs). At that time, this discovery had a great impact on the field because mutations driving the development of PMF had yet to be discovered. Later studies identified that PMF, as the others MPNs, is associated with mutations activating the thrombopoietin/JAK2 axis raising great hope that JAK inhibitors may be effective to treat the disease. Unfortunately, ruxolitinib, the JAK1/2 inhibitor approved by FDA and EMEA for PMF, ameliorates symptoms but does not improve the natural course of the disease, and the cure of PMF is still an unmet clinical need. Although GATA1 is not mutated in PMF, reduced GATA1 content in megakaryocytes as a consequence of ribosomal deficiency is a hallmark of myelofibrosis (both in humans and mouse models) and, in fact, a driving event in the disease. Conversely, mice carrying the hypomorphic Gata1low mutation express an activated TPO/JAK2 pathway and partially respond to JAK inhibitors in a fashion similar to PMF patients (reduction of spleen size but limited improvement of the natural history of the disease). These observations cross-validated Gata1low mice as a bona fide animal model for PMF and prompted the use of this model to identify abnormalities that might be targeted to cure the disease. We will summarize here data generated in Gata1low mice indicating that the TGF-ß/P-selectin axis is abnormal in PMF and represents a novel target for its treatment.

Topological complexity of photons' paths in biological tissues.

In the present contribution, three means of measuring the geometrical and topological complexity of photons' paths in random media are proposed. This is realized by investigating the behavior of the average crossing number, the mean writhe, and the minimal crossing number of photons' paths generated by Monte Carlo (MC) simulations, for different sets of optical parameters. It is observed that the complexity of the photons' paths increases for increasing light source/detector spacing, and that highly "knotted" paths are formed. Due to the particular rules utilized to generate the MC photons' paths, the present results may have an interest not only for the biomedical optics community, but also from a pure mathematical point of view.

P3 solution for the total steady-state and time-resolved reflectance and transmittance from a turbid slab.

In this paper, we derive some explicit analytical solutions to the P3 equations for the slab geometry that is illuminated by a collimated plane source. The resulting expressions for the total reflectance and transmittance are compared with the corresponding transport theory solution predicted by the Monte Carlo method. Further, for the special case of a non-absorbing anisotropically scattering slab, simple and accurate expressions in the P1 approximation are obtained, yielding for optically thick slabs, the typical behavior of Ohm's law. In view of the time domain, we present an alternative method to the classical frequency-domain approach avoiding the use of complex numbers.

Heuristic model for ballistic photon detection in collimated transmittance measurements.

An heuristic model for ballistic photon detection in continuous-wave measurements of collimated transmittance through a slab is presented. The model is based on the small angle approximation and the diffusion equation and covers all the ranges of optical thicknesses of the slab from the ballistic to the diffusive regime. The performances of the model have been studied by means of comparisons with the results of gold standard Monte Carlo simulations for a wide range of optical thicknesses and two types of scattering functions. For a non-absorbing slab and field of view of the receiver less than 3° the model shows errors less than 15% for any value of the optical thickness. Even for an albedo value of 0.9, and field of view of the receiver less than 3° the model shows errors less than 20%. These results have been verified for a large set of scattering functions based on the Henyey-Greenstein model and Mie theory for spherical scatterers. The latter has also been used to simulate the scattering function of Intralipid, a diffusive material widely used as reference standard for tissue simulating phantoms. The proposed model represents an effective improvement compared to the existing literature.

Random laser based method for direct measurement of scattering properties.

Optical sensing is a very important method for investigating different kinds of samples. Recently, we proposed a new kind of optical sensor based on random lasing [ Sci. Rep.6, 35225 (2016)], that couples the advantages of stimulated emission in detecting small variations on scattering properties of a sensed material, to the needs of no alteration of the sample under investigation. Here, we present a method to achieve a quantitative measurement of the scattering properties of a material. The results on samples of calibrated microspheres show a dependence of the peak intensity of the emission spectrum on the transport mean free path of the light within the sample, whatever the dimension (down to ≈100 nm of particle diameter) and the concentration of scatterers dispersed in the sensed material. A direct and fast measurement of the scattering properties is obtained by calibration with a well-known and inexpensive reference medium.

Generalized time-independent correlation transport equation with static background: influence of anomalous transport on the field autocorrelation function.

A generalized time-independent correlation transport equation (GCTE) is proposed for the field autocorrelation function. The GCTE generalizes various models for anomalous transport of photons and takes into account the possible presence of a static background. In a tutorial example, the GCTE is solved for a homogeneous semi-infinite medium in reflectance configuration through Monte Carlo simulations. The chosen anomalous photon transport model also includes the classic and the "generalized" Lambert-Beer's law (depending on the choice of parameters). A numerical algorithm allowing generation of the related anomalous random photon steps is also given. The clear influence of anomalous transport on the field autocorrelation function is shown and discussed for the proposed specific examples by comparing the general results with the classical case (Lambert-Beer's law).

Frequency offset Raman spectroscopy (FORS) for depth probing of diffusive media.

We present a new technique, frequency offset Raman spectroscopy (FORS), to probe Raman spectra of diffusive media in depth. The proposed methodology obtains depth sensitivity exploiting changes in optical properties (absorption and scattering) with excitation wavelengths. The approach was demonstrated experimentally on a two-layer tissue phantom and compared with the already consolidated spatially offset Raman spectroscopy (SORS) technique. FORS attains a similar enhancement of signal from deep layers as SORS, namely 2.81 against 2.62, while the combined hybrid FORS-SORS approach leads to a markedly higher 6.0 enhancement. Differences and analogies between FORS and SORS are discussed, suggesting FORS as an additional or complementary approach for probing heterogeneous media such as biological tissues in depth.

P-Selectin Sustains Extramedullary Hematopoiesis in the Gata1 low Model of Myelofibrosis.

Splenomegaly is a major manifestation of primary myelofibrosis (PMF) contributing to clinical symptoms and hematologic abnormalities. The spleen from PMF patients contains increased numbers of hematopoietic stem cells (HSC) and megakaryocytes (MK). These MK express high levels of P-selectin (P-sel) that, by triggering neutrophil emperipolesis, may cause TGF-ß release and disease progression. This hypothesis was tested by deleting the P-sel gene in the myelofibrosis mouse model carrying the hypomorphic Gata1(low) mutation that induces megakaryocyte abnormalities that recapitulate those observed in PMF. P-sel(null) Gata1(low) mice survived splenectomy and lived 3 months longer than P-sel(WT) Gata1(low) littermates and expressed limited fibrosis and osteosclerosis in the marrow or splenomegaly. Furthermore, deletion of P-sel disrupted megakaryocyte/neutrophil interactions in spleen, reduced TGF-ß content, and corrected the HSC distribution that in Gata1(low) mice, as in PMF patients, is abnormally expanded in spleen. Conversely, pharmacological inhibition of TGF-ß reduced P-sel expression in MK and corrected HSC distribution. Spleens, but not marrow, of Gata1(low) mice contained numerous cKIT(pos) activated fibrocytes, probably of dendritic cell origin, whose membrane protrusions interacted with MK establishing niches hosting immature cKIT(pos) hematopoietic cells. These activated fibrocytes were not detected in spleens from P-sel(null) Gata1(low) or TGF-ß-inhibited Gata1(low) littermates and were observed in spleen, but not in marrow, from PMF patients. Therefore, in Gata1(low) mice, and possibly in PMF, abnormal P-sel expression in MK may mediate the pathological cell interactions that increase TGF-ß content in MK and favor establishment of a microenvironment that supports myelofibrosis-related HSC in spleen.

Study on the mathematical relationship existing between single-photon laser-Doppler flowmetry and diffuse correlation spectroscopy with static background.

In the present contribution, the theoretical relationship existing between the blood flow index measured by diffuse correlation spectroscopy and single-photon laser-Doppler flowmetry (SP-LDF) is investigated. A specific mathematical description that accounts for the properties of single-photon detectors for SP-LDP was developed. Static background has also been considered and, to the best of our knowledge, this has never been included before in SP-LDF analytical theories. The comparisons were realized for two SP-LDF implementations: for "classical" and "fast" algorithms. "Classical" SP-LDF is not sensitive to small changes on the number of detected speckles and coherence length of the laser, usually described by a unique parameter "beta." This is a strong point when assessing blood flow index, e.g., in humans, where "beta" is particularly difficult to be determined in real time. The proposed theory may be utilized, e.g., to investigate other SP-LDF setups and optical/physiological parameter ranges or, generally, to optimize real SP-LDF instrumentation.

Derivation of the correlation diffusion equation with static background and analytical solutions.

A new correlation diffusion equation has been derived from a correlation transport equation allowing one to take into account the presence of moving scatterers and static background. Solutions for the reflectance from a semi-infinite medium have been obtained (point-like and ring detectors). The solutions have been tested by comparisons with "gold standard" Monte Carlo (MC) simulations. These formulas suitably describe the electric field autocorrelation function, for Brownian or random movement of the scatterers, even in the case where the probability for a photon to interact with a moving scatterer is very low. The proposed analytical models and the MC simulations show that the "classical" model, often used in diffuse correlation spectroscopy, underestimates the normalized field autocorrelation function for increasing correlation times.

Time-domain Raman analytical forward solvers.

A set of time-domain analytical forward solvers for Raman signals detected from homogeneous diffusive media is presented. The time-domain solvers have been developed for two geometries: the parallelepiped and the finite cylinder. The potential presence of a background fluorescence emission, contaminating the Raman signal, has also been taken into account. All the solvers have been obtained as solutions of the time dependent diffusion equation. The validation of the solvers has been performed by means of comparisons with the results of "gold standard" Monte Carlo simulations. These forward solvers provide an accurate tool to explore the information content encoded in the time-resolved Raman measurements.

Analytical solution of the correlation transport equation with static background: beyond diffuse correlation spectroscopy.

The correlation transport equation (CTE) is the natural generalization of the theory for diffusion correlation spectroscopy and represents a more precise model when dealing with measurements of particle movement in fluids or red blood cell flow in biological tissues. Unfortunately, the CTE is not methodically used due to the complexity of finding solutions. It is shown that actually a very simple modification of the theory/software for the solution of the radiative transport equation allows one to obtain exact solutions of the CTE. The presence of a static background is also taken into account and its influence on the CTE solutions is discussed. The proposed approach permits one to easily work beyond the diffusion regime and potentially for any optical and/or physiological value. The validity of the approach is demonstrated by using "gold standard" Monte Carlo simulations.

Probability density function of the electric field in diffuse correlation spectroscopy of human bone in vivo.

Diffuse correlation spectroscopy (DCS) is the technique of choice for non-invasive assessments of human bone blood flow. However, DCS classical algorithms are based on the fundamental assumption that the electric field of the light reaching the DCS photodetector is a zero-mean complex Gaussian variable. The non-validity of this hypothesis might produce inaccurate blood flow estimations. It is shown that for the human tibia, the "Gaussian hypothesis" holds for interoptode distances ≥20 mm. This lower boundary seems to depend on the type of investigated tissue.

Characterization of the TGF-ß1 signaling abnormalities in the Gata1low mouse model of myelofibrosis.

Primary myelofibrosis (PMF) is characterized by fibrosis, ineffective hematopoiesis in marrow, and hematopoiesis in extramedullary sites and is associated with abnormal megakaryocyte (MK) development and increased transforming growth factor (TGF)-ß1 release. To clarify the role of TGF-ß1 in the pathogenesis of this disease, the TGF-ß1 signaling pathway of marrow and spleen of the Gata1(low) mouse model of myelofibrosis (MF) was profiled and the consequences of inhibition of TGF-ß1 signaling on disease manifestations determined. The expression of 20 genes in marrow and 36 genes in spleen of Gata1(low) mice was altered. David-pathway analyses identified alterations of TGF-ß1, Hedgehog, and p53 signaling in marrow and spleen and of mammalian target of rapamycin (mTOR) in spleen only and predicted that these alterations would induce consequences consistent with the Gata1(low) phenotype (increased apoptosis and G1 arrest both in marrow and spleen and increased osteoblast differentiation and reduced ubiquitin-mediated proteolysis in marrow only). Inhibition of TGF-ß1 signaling normalized the expression of p53-related genes, restoring hematopoiesis and MK development and reducing fibrosis, neovascularization, and osteogenesis in marrow. It also normalized p53/mTOR/Hedgehog-related genes in spleen, reducing extramedullary hematopoiesis. These data identify altered expression signatures of TGF-ß1 signaling that may be responsible for MF in Gata1(low) mice and may represent additional targets for therapeutic intervention in PMF.