Homaira Rahimi

In vivo quantification of lymph viscosity and pressure in lymphatic vessels and draining lymph nodes of arthritic joints in mice

Previously, it was found that the popliteal lymph node (PLN) enlarges during the pre‐arthritic ‘expanding’ phase, and then ‘collapses’ with adjacent knee flare and is associated with the loss of the intrinsic lymphatic pulse. However, the mechanisms responsible are unknown and we therefore developed in vivo methods to measure lymph viscosity, lymphatic pumping pressure (LPP) in the lymphatic vessels afferent to the PLN, and lymph node pressure (LNP). Multiphoton fluorescence recovery after photobleaching (MP‐FRAP) was used to calculate lymph viscosity and speed; no difference was found among mice with wild‐type (WT), expanding or collapsed PLN in lymph viscosity, but lymph speed was found to be decreased in mice with collapsed PLN compared to WT and expanding PLN mice. LPP was measured indirectly by slowly releasing a pressurized cuff occluding ICG fluorescent dye; we found that mice with expanding PLN exhibit a higher LPP compared to WT and mice with collapsed PLN show an extremely low LPP. Direct measurement of LNP demonstrated a decrease in expanding PLN versus WT pressure, which dramatically increased in collapsed PLN. The decrease in lymphatic flow and loss of LPP during PLN collapse are consistent with decreased drainage from the joint during arthritic flare, and validate these biomarkers of rheumatoid arthritis progression and possibly other chronic inflammatory conditions

Childhood Arthritis and Rheumatology Research Alliance Consensus Clinical Treatment Plans for Juvenile Dermatomyositis with Persistent Skin Rash

Objective. Juvenile dermatomyositis (JDM) is the most common form of idiopathic inflammatory myopathy in children. While outcomes are generally thought to be good, persistence of skin rash is a common problem. The goal of this study was to describe the development of clinical treatment plans (CTP) for children with JDM characterized by persistent skin rash despite complete resolution of muscle involvement. Methods. The Childhood Arthritis and Rheumatology Research Alliance, a North American consortium of pediatric rheumatologists and other healthcare providers, used a combination of Delphi surveys and nominal group consensus meetings to develop CTP that reflected consensus on typical treatments for patients with JDM with persistent skin rash. Results. Consensus was reached on patient characteristics and outcome assessment. Patients should have previously received corticosteroids and methotrexate (MTX). Three consensus treatment plans were developed. Plan A added intravenous immunoglobulin (IVIG) if it was not already being used. Plan B added mycophenolate mofetil, while Plan C added cyclosporine. Continuation of previous treatments, including corticosteroids, MTX, and IVIG, was permitted in plans B and C. Conclusion. Three consensus CTP were developed for use in children with JDM and persistent skin rash despite complete resolution of muscle disease. These CTP reflect typical treatment approaches and are not to be considered treatment recommendations or standard of care. Using prospective data collection and statistical methods to account for nonrandom treatment assignment, it is expected that these CTP will be used to allow treatment comparisons, and ultimately determine the best treatment for these patients.

Power Doppler Ultrasound Phenotyping of Expanding versus Collapsed Popliteal Lymph Nodes in Murine Inflammatory Arthritis

Rheumatoid arthritis is a chronic inflammatory disease manifested by episodic flares in affected joints that are challenging to predict and treat. Longitudinal contrast enhanced-MRI (CE-MRI) of inflammatory arthritis in tumor necrosis factor-transgenic (TNF-Tg) mice has demonstrated that popliteal lymph nodes (PLN) increase in volume and contrast enhancement during the pre-arthritic “expanding” phase of the disease, and then suddenly “collapse” during knee flare. Given the potential of this biomarker of arthritic flare, we aimed to develop a more cost-effective means of phenotyping PLN using ultrasound (US) imaging. Initially we attempted to recapitulate CE-MRI of PLN with subcutaneous footpad injection of US microbubbles (DEFINITY®). While this approach allowed for phenotyping via quantification of lymphatic sinuses in PLN, which showed a dramatic decrease in collapsed PLN versus expanding or wild-type (WT) PLN, electron microscopy demonstrated that DEFINITY® injection also resulted in destruction of the lymphatic vessels afferent to the PLN. In contrast, Power Doppler (PD) US is innocuous to and efficiently quantifies blood flow within PLN of WT and TNF-Tg mice. PD-US demonstrated that expanding PLN have a significantly higher normalized PD volume (NPDV) versus collapsed PLN (0.553±0.007 vs. 0.008±0.003; p<0.05). Moreover, we define the upper (>0.030) and lower (<0.016) quartile NPDVs in this cohort of mice, which serve as conservative thresholds to phenotype PLN as expanding and collapsed, respectively. Interestingly, of the 12 PLN phenotyped by the two methods, there was disagreement in 4 cases in which they were determined to be expanding by CE-MRI and collapsed by PD-US. Since the adjacent knee had evidence of synovitis in all 4 cases, we concluded that the PD-US phenotyping was correct, and that this approach is currently the safest and most cost-effective in vivo approach to phenotype murine PLN as a biomarker of arthritic flare.

Altered Bone Biology in Psoriatic Arthritis

Psoriatic arthritis (PsA) is characterized by focal bone erosions mediated by osteoclasts at the bone–pannus junction. The bulk of research over the past decade has centered on mechanisms that underlie osteoclastogenesis along with new insights into osteoimmunology; however, recent advances that focus on steps that lead to new bone formation are beginning to emerge. New revelations about bone formation may have direct relevance to PsA given the presence of enthesophytes, syndesmophytes, and bony ankylosis frequently observed in patients with this disorder. In this review, we discuss current developments in the pathogenesis of new bone formation, novel imaging approaches to study bone remodeling and highlight innovative approaches to study the effect of inflammation on bone. Lastly, we discuss promising therapies that target joint inflammation and osteitis with the potential to mediate pathologic bone formation.

Brief Report: Treatment of Tumor Necrosis Factor–Transgenic Mice With Anti–Tumor Necrosis Factor Restores Lymphatic Contractions, Repairs Lymphatic Vessels, and May Increase Monocyte/Macrophage Egress

Recent studies have demonstrated that there is an inverse relationship between lymphatic egress and inflammatory arthritis in affected joints. As a model, tumor necrosis factor (TNF)–transgenic mice develop advanced arthritis following draining lymph node (LN) collapse, and loss of lymphatic contractions downstream of inflamed joints. It is unknown if these lymphatic deficits are reversible. This study was undertaken to test the hypothesis that anti‐TNF therapy reduces advanced erosive inflammatory arthritis, associated with restoration of lymphatic contractions, repair of damaged lymphatic vessels, and evidence of increased monocyte egress.

Validation of Power Doppler Versus Contrast-Enhanced Magnetic Resonance Imaging Quantification of Joint Inflammation in Murine Inflammatory Arthritis

Contrast‐enhancement magnetic resonance imaging (CE‐MRI) of synovial volume is the radiographic gold standard to quantify joint inflammation; however, cost limits its use. Therefore, we examined if power Doppler‐ultrasound (PD‐US) outcomes of synovitis in tumor necrosis factor transgenic (TNF‐Tg) mice correlate with CE‐MRI. TNF‐Tg mice underwent PD‐US of their knees to measure the joint space volume (JSV) and power Doppler volume (PDV), and the results were correlated with synovial volume determined by CE‐MRI. Immunohistochemistry for CD31 was performed to corroborate the PD signal. Synovial volume strongly correlated with both JSV and PDV (p < 0.01). CD31+ blood vessels were observed in inflamed synovium proximal to the joint surface, which corresponded to areas of intense PD signals. JSV and PDV are valid measures of joint inflammation that correlate with synovial volume determined by CE‐MRI and are associated with vascularity. Given the emergence of PD‐US as a nonquantitative outcome of joint inflammation, we find JSV and PDV to be feasible and highly cost‐effective for longitudinal studies in animal models. Furthermore, given the increasing use of PD‐US in standard clinical practice, JSV and PDV could be translated to better quantify joint flare and response to therapy in patients with rheumatoid arthritis (RA).

Treatment of TNF‐Tg Mice with Anti‐TNF Restores Lymphatic Contraction, Repairs Lymphatic Vessels, and May Increase Monocyte/Macrophage Egress

Recent studies have demonstrated that there is an inverse relationship between lymphatic egress and inflammatory arthritis in affected joints. As a model, tumor necrosis factor (TNF)–transgenic mice develop advanced arthritis following draining lymph node (LN) collapse, and loss of lymphatic contractions downstream of inflamed joints. It is unknown if these lymphatic deficits are reversible. This study was undertaken to test the hypothesis that anti‐TNF therapy reduces advanced erosive inflammatory arthritis, associated with restoration of lymphatic contractions, repair of damaged lymphatic vessels, and evidence of increased monocyte egress.

Increased numbers of CD23+CD21hi Bin‐like B cells in human reactive and rheumatoid arthritis lymph nodes

A unique population of CD23+ CD21high B cells in inflamed nodes (Bin) has been shown to accumulate in lymph nodes (LNs) draining inflamed joints of TNF‐transgenic (TNF‐tg) mice. Bin cells contribute to arthritis flare in mice by distorting node architecture and hampering lymphatic flow, but their existence in human inflamed LNs has not yet been described. Here, we report the characterization of resident B‐cell populations in fresh popliteal lymph nodes (PLNs) from patients with severe lower limb diseases (non‐RA) and rheumatoid arthritis (RA) patients, and from banked, cryopreserved reactive and normal human LN single cell suspension samples. Bin‐like B cells were shown to be significantly increased in reactive LNs, and strikingly elevated (>30% of total) in RA samples. Histopathology and immunofluorescence analyses were consistent with B follicular hyperplasia and histological alterations in RA vs. non‐RA PLNs. This is the first description of Bin‐like B cells in human inflamed LNs. Consistent with published mouse data, this population appears to be associated with inflammatory arthritis and distortion of LN architecture. Further analyses are necessary to assess the role of CD23+CD21hi Bin‐like B cells in RA pathogenesis and arthritic flare.

Validation of 3‐dimensional ultrasound versus magnetic resonance imaging quantification of popliteal lymph node volume as a biomarker of erosive inflammatory arthritis in mice

Draining lymph node (LN) enlargement has long been recognized as a hallmark of joint inflammation in rheumatoid arthritis (RA), and can be quantified with magnetic resonance imaging (MRI) (1). The importance of this outcome measure as a biomarker of inflammatory-erosive arthritis initiation, progression, and response to therapy has recently been demonstrated in murine models (2–5). Despite its potential value, this approach has not gained broad acceptance due to its very high costs (money, time and labor), and limited access to MRI machines. Thus, investigators have been evaluating ultrasound (US) as a more practical and cost-effective method to study LN in animal models (6), and RA patients (7). Based on these promising results, we aimed to validate in vivo US volume measurement of popliteal lymph nodes (PLN) in TNF-transgenic (TNF-Tg) mice (8) with varying degrees of arthritis by comparing the results obtained using both imaging modalities. A total of 16 PLNs from 3 to 8-month-old heterozygous TNF-Tg mice (3647 line in a C57B6 background) were anesthetized with intraperitoneal ketamine (60 mg/kg) and xylazine (4mg/kg), scanned by MRI, and their volumes were determined with Amira software (Visage Imaging, Inc., San Diego, CA) as described previously (3). On the following day, these PLNs were imaged with a high-resolution small-animal ultrasound system (VisualSonics 770 with 704 scanhead). Each mouse was anesthetized with ~2% isoflurane in oxygen. Hair was removed from ankles to hips using a depilatory cream. The mouse was placed in the supine position on the 40°C heated imaging platform with paws taped to surface electrodes for heart rate monitoring and respiratory rate synchronization (Fig. 1A). The PLN was identified in brightness-mode (B-mode) by adjusting the scanhead up or down to position the PLN at the plane of focus (red arrow in Fig. 1B), and then scanned in three dimension-mode (3D-mode) with a step size of 0.032 mm. The 3D US image data were used to quantify PLN volume by manual segmentation of the lymph node and surrounding fat pads. The mean signal intensity of the fat pad (FPsi) was computed using the TissueStatistics module. To eliminate the fat pad from the node material, selected areas in which the signal intensity was over FPsi were subtracted and any resultant empty inclusions (“holes”) within the node were filled. However, holes on the surface cannot be filled by this approach. The SurfaceGen module was used to arrange the labeled pixels as a bounded surface for subsequent 3D visualization and volumetric quantification with the SurfaceView (Fig. 1C) and TissueStatistics modules. A linear regression analysis was performed on the volumes generated from both imaging modalities (Fig. 1D). We also determined the intra and inter-observer reliability of our US PLN volume measurement, which showed insignificant variability (p = 0.8399 and 0.8096 respectively). Figure 1 Strong correlation between PLN volumes determined by MRI vs. US US proved to be a very facile approach to assess murine PLN (Fig. 1B), since they are readily identified in B-mode after locating the triangular fat pad. A strong relationship between PLN volumes measured by MRI and US was found using a linear regression model (R2 = 0.844, P<0.0001) (Fig. 1D). However, vertical placement of PLN in US image is a potential source of variability, perhaps as much as 10 percent (see below), and may result from mechanical compression with the scanhead. To reduce this variation, a lower frequency scanhead could be used resulting in a greater focal depth and distance from the head. In addition, the scanhead position should be adjusted so that the PLN is centered consistently in the plane of focus (Fig. 1B). Of note is that smaller PLN are less susceptible to scanhead compression error, as suggested by the stronger linear relationship with the volume obtained from MRI. To give a broader illustration of the correlation between MRI and US measurements, 3 PLN were chosen to represent the smaller, middle and larger PLN, and their 3D rendered images generated by MRI and US are presented (Fig. 1E–J). One surprise of our study was that the slope was not 1.0, despite the strong correlation between measurements on the same node across animals on the two instruments (Fig. 1D). To test the hypothesis that we might be compressing the node during US imaging, we varied the vertical position of the scanhead in an attempt to compress the node. In a representative test, the PLN showed a volume of 6.86mm3 at a depth of 7mm, and a volume of 6.39mm3 at a depth of 5mm, resulting in a ~10 % difference in node volume. This is not large enough to account for the differences we observed (slope = 1.45). Thus, other factors must also contribute to the differences that we observed, and accuracy of the absolute volume measurements attainable with these non-invasive approaches remains a limitation. Another error with the US measurement is the roughened surface (Figure 1F,H,J), which occurs due to our inability to fill surface holes. This should be addressable in the future with the evolution of superior surface rendering software applications. However, since the primary outcome measure of this biomarker is to predict RA progression by PLN volume enlargement, we conclude that larger PLN imaged in MRI will also be larger during ultrasound imaging. The three largest and the three smallest nodes (Figure 1D) clearly differ from one another, and the rank order of size derived from each modality is the same for these six observations. The nodes in the middle of the range are closely bunched and may not differ significantly from one another in each group. Although palpable draining LN have long been recognized as a symptom of RA, their value as a quantitative biomarker of disease initiation, arthritic flare and response to therapy has only recently been appreciated (7). However, if this biomarker is to be broadly utilized, it needs to be assessed by practical means such as US, which can be readily performed during the office visit. For this reason, US imaging has recently been evaluated as an alternative to MRI to assess various musculoskeletal conditions. In some cases, such as detecting psoriatic arthritis of fingers and toes in patients with psoriasis (9), and detection of bone erosions in gouty arthritis (10), US has been shown to be just as effective as MRI. However, in other cases such as predicting the development of RA from undifferentiated peripheral inflammatory arthritis, MRI assessment of bone edema, synovitis and erosion pattern proved to be more useful (11). In summary, here we demonstrate that US is comparable to MR imaging for determining relative PLN volume in mice with inflammatory arthritis. Since this can be performed at less than 10% of the financial cost, we find that US is a quick, inexpensive, and reliable method to interrogate this biomarker of RA pathogenesis and response to therapy.

Lymphatic imaging to assess rheumatoid flare: mechanistic insights and biomarker potential

AbstractProliferation of draining lymphatic vessels coupled with dynamic changes in lymph node volume and flow are characteristic features in rheumatoid arthritis (RA). Furthermore, impaired lymph egress from inflamed synovium is associated with joint flare in murine models of inflammatory-erosive arthritis. Unfortunately, advances towards a greater understanding of lymphatic changes in RA pathogenesis have been slow due to the absence of outcome measures to quantify lymphatic function in vivo. While lymphoscintigraphy is the current standard to assess lymphedema and sentinel lymph nodes in cancer patients, its sensitivity and specificity are inadequate to study lymphatics in RA. The emergence of high-resolution MRI, power Doppler ultrasound, and near-infrared imaging that permits real-time quantification of lymphatic function in animal models has been a major advance, and these techniques have produced a new paradigm of altered lymphatic function that underlies both acute arthritic flare and chronic inflammation. In acute flare, lymphatic drainage increases several fold, whereas no lymphatic contractions are detected in lymph vessels draining chronic arthritic joints. Moreover, these outcomes are now being adapted to study lymphatics in RA towards the development of novel biomarkers of arthritic flare and the discovery of new therapeutic targets. In particular, interventions that directly increase lymphatic egress from diseased joints by opening collateral lymphatic vessels, and that restore lymphatic vessel contractions, provide novel therapeutic approaches with potential for minimal toxicity and immunosuppression. To summarize the origins of this field, recent advances, and future directions, we herein review: current knowledge of lymphatics in RA based on classic literature; new in-vivo imaging modalities that have elucidated how lymphatics modulate acute versus chronic joint inflammation in murine models; and how these preclinical outcome measures are being translated to study lymphatic function in RA inflammation and how effective RA therapies alter lymphatic flow and lymph nodes draining flaring joints. Trial registration: ClinicalTrials.gov NCT02680067. Registered 7 December 2015; ClinicalTrials.gov NCT01098201. Registered 30 March 2010; and ClinicalTrials.gov NCT01083563. Registered 8 March 2010.