Hypoxia causes the expression of signaling molecules which regulate cell division, lead to angiogenesis, and further, in the tumor microenvironment, promote resistance to chemotherapy and radiotherapy, and induce metastasis. Photoacoustic imaging (PAI) takes advantage of unique absorption characteristics of chromophores in tissues and provides the opportunity to construct images with a high degree of spatial and temporal resolution. In this review, we discuss the physiologic characteristics of tumor hypoxia, and current applications of PAI using endogenous (label free imaging) and exogenous (organic and inorganic) contrast agents. Features of various methods in terms of their efficacy for determining physiologic and proteomic phenomena are analyzed. This review demonstrates that PAI has the potential to understand tumor growth and metastasis development through measurement of regulatory molecule concentrations, oxygen gradients, and vascular distribution. EMT bestows tumor cells with more invasive and migratory phenotypes for enhanced metastases Tumor hypoxia is a hallmark of the tumor microenvironment and is defined as a lack of oxygenation to the tumor tissue, due to insufficient blood supply Development and approval of a method which can accurately investigate and assess tumor hypoxia will lead to more appropriate treatment plans - which will ultimately lead to greater survival rates and reductions in cancer metastases. Existing investigations of tumor hypoxia can be categorized into two approaches: (1) determination of proteomic and genomic transformation of tumor cells in response to hypoxia, and 2) evaluation of physiological hemodynamic and/or metabolic alterations due to the onset of hypoxia , concentration of endogenous chromophores (i.e. oxy- and deoxyhemoglobin), tissue perfusion based on blood flow measurement, oxygen saturation concentration (sO2), metabolic rate of oxygenation (MrO2) and uptake of exogenous biomarkers or radiolabeled hypoxia reporters Next we consider determining and assessing tumor hypoxia through evaluation of local physiological hemodynamic and/or metabolic alterations that can be analyzed through different technologies: determining partial pressure of oxygen . Moreover, the probe is invasive and is limited to surface tumors 2 values can also be determined by using oxygen-sensitive electron paramagnetic resonance (EPR) probes where spectral band corresponding to the probe signal correlates with oxygen concentration 19F-magnetic resonance spectroscopy (MRS) and Overhauser-enhanced MRI (OMRI) are two magnetic resonance-based imaging modalities capable of providing absolute pO2.Values of pO2) nanoparticles to enhance contrast between healthy and malignant tumor tissue 2 can adequately diagnose hypoxic regions of tumor, may enable predictions of responses to radiotherapy and immune checkpoint inhibitors 2 and loaded exosomes) are severely toxic for patients, so far limiting this method to research studies There are several imaging modalities used to study tumor hypoxia based on the evaluation of indirect hemodynamic changes. There are two types of MRI that have been developed for indirect assessment of tumor hypoxia, dynamic contrast enhanced-MRI (DCE-MRI) and blood oxygenation level-dependent MRI (BOLD-MRI) 2) to quantify a ratio of Hb/HbO2. Based on the differential feedback of these two values, sO2 is calculated and an estimated pO2 value can be determined based on the oxygen dissociation curve. However, due to the inherent optical diffusion limit, this method provides low spatial resolution, and has a poor depth penetration In vivo bioluminescence imaging 2Modalities such as positron emission tomography (PET) are minimally invasive techniques based on the uptake of intravenously administered radiolabeled reporters to detect tumor hypoxia. However, PET is not effective for determining hypoxia in a clinical setting due to impractical temporal resolution that does not allow real time imaging of tissue hypoxia Fluorescent microscopy is an established imaging modality and a plethora of fluorescent probes able to detect hypoxia through bioactivity of nitroreductase (NTR) and other mechanisms exist. The difficulty of using fluorescent microscopy lies with the poor penetration of optical microscopy in general, requiring highly luminescent signals to penetrate through highly attenuating biological tissue. For example, the probe GCTPOC-HY has a 130-fold fluorescent enhancement, yet when applied to monitor NTR via two-photon microscopy it only demonstrated a penetration depth of 70 μm Table Photoacoustic imaging (PAI) is an emerging technique which couples optical sensitivity with acoustic detection capabilities and has been used extensively in research  following light absorption can be written as:In PAI, the imaging target  is optically excited using a pulsed laser, leading to a transient temperature increase and resulting in a thermoelastic expansion of the absorber followed by emission of acoustic pressure waves. This initial pressure rise  can be represented by the thermal expansion coefficient , the compressibility , and the speed of sound  and can further be expanded in terms of the mass density  and specific heat capacity at constant volume . The absorbed optical energy is represented as the product of the absorption coefficient  and the fluence at the local absorption point . The emitted acoustic pressure waves from the absorber are propagated through the adjacent tissue layers and eventually detected by ultrasound (US) transducers placed on the skin. These acoustic waves induced from optical energy absorption, also known as PAsignal, are used to generate real-time, high-resolution images representing functional and molecular characteristic distributions of tissue chromophores at higher depths than pure optical imaging. Therefore, PAI combines the advantages of the unique optical absorption characteristics of tissue chromophores with the higher penetration depth of ultrasound Where the Grüneisen coefficient, 2) and deoxy-hemoglobin (HbR)) enables spectroscopic or multi-wavelength imaging techniques to denote functional characteristics of the tumor region 2 and HbR have different wavelength-dependent optical absorption properties, which allows spectroscopic PAI to differentiate between arteries and veins. Furthermore, optical absorption by HbO2 and HbR at two or more wavelengths , can be used to assess total hemoglobin concentration (tHb) and, therefore, derive, blood oxygen saturation (sO2) ) of HbO2 and HbR are known at specific wavelengths and HbO2 and HbR concentrations (HbOC2 and HbC) at specific image locations can be determined from PA signal intensity. Total Hb can be calculated by , and sO2 can be calculated as follows:The physiological and pathological status of the tumor  can be well defined based on the intensity contrast (compared to background) in PA images generated by the absorbing chromophores within and around the tumor region in vivo oxygenation imaging at a relatively low cost The hemoglobin-based oxygen saturation measurement is referred to as “label-free” PAI 2 has been successfully quantified from PA imaging based on the following equation:In ε denotes the oxygen binding capacity of hemoglobin and is usually considered a constant, A denotes cross-sectional area,  is the average blood flow speed (in or out), and W is the weight of the region of interest. These parameters are required to be measured locally. Anatomic parameters i.e., weight and area are quantified from the structural information extracted from PA image and fluid-dynamic parameter i.e., flow speed can be estimated on the basis of PA Doppler bandwidth broadening of the PA signal induced by circulating red blood cells. Another physiological assessment of hypoxia can be implemented by measuring oxygen extraction factor (OEF) as follows Where 2 in tissue 2 concentration, the contrast agent half-life is reduced, decreasing the PA signal intensity. An exponential fitting function is applied to the PA signal intensity decay to calculate pO2PA contrast can also be enhanced by exogenous contrast agents (organic or inorganic) that have distinct and tunable absorption spectra in the near-infrared (NIR) optical window (600-1100 nm) Although PAI has significant benefits in a research setting, realization of this modality is also practical in a clinical setting in vivo or in vitro experiments, utilized PAI specifically as well utilized animal cancer models. The exclusion criteria of this paper were articles which utilized PAI to determine the efficacy of hypoxia targeted drug delivery, papers which used modalities aside from PAI to investigate tumor hypoxia such as bioluminescence imaging, fluorescence imaging, and PET.Here, we reviewed various approaches for evaluating biomarkers of tissue hypoxia, such as oxygen saturation, partial pressure of oxygen, and metabolic rate of oxygen consumption. The assessments of hypoxia approaches were classified as follows: (1) from physiological changes using endogenous, label-free biomarkers; and (2) from physiological/ molecular alterations using exogenous contrast agents by either (a) an estimation of physiological changes using exogenous contrast agents or (b) an estimation of oxygen sensitive proteomic transformation/exogenous contrast agent activation. Journal articles relevant to this review were gathered by conducting an online search using PubMed. The goal of this review is to study relevant articles which used PAI to assess tumor hypoxia. This initial search utilized the keywords: 'photoacoustic imaging', 'hypoxia', 'tumor hypoxia' and 'photoacoustic technologies'. Furthermore, the following MeSH terms were also used: 'contrast agents', 'hypoxia' and 'cancer'. This search yielded 174 results, of which 26 papers were selected for this review. The inclusion criteria for conducting the review were the articles which utilized 2) values were determined as explained in section 2. Label free imaging has been shown to adequately measure oxygenation in 2, and MrO2 have been determined via label free PAI 2 at early stages followed by a gradual decline in MrO2 starting from day 14. This phenomenon explains the relative gradual tumor growth as well as tumor core necrosis.Label free imaging provides the advantage of using the unique absorption spectra of endogenous contrast agents, particularly, oxygenated and deoxygenated hemoglobin 2 levels are inversely correlated to tumor volume 2 levels decrease 2 as the tumor grows Moreover, label free imaging has been able to document changes in local oxygen saturation as a tumor progresses Label free imaging was used to investigate tumor hypoxia in multiple types of cancers In addition, label free imaging can potentially be used as a cancer risk assessment tool Furthermore, label free imaging may be useful in predicting tumor recurrence. Murine model of glioblastoma implanted in hind leg, receiving photodynamic therapy (PDT), was imaged with label free PAI to determine treatment efficacy and risk of tumor recurrence 22 map was generated pre- and post-PDT using MSOT. Interestingly, 40 minutes post-PDT the entire tumor center became hypoxic, and 60 minutes post-PDT the whole tumor mass was hypoxic showing a 60% drop in sO2. However, oxygenation levels returned to baseline 48 hours post-PDT, whereas the control group showed consistent sO2 levels over the course of 48 hours   to determine the impact of vascular targeted photodynamic therapy on sOsee Fig. . These sIn addition to simply reporting oxygenation levels, PAI can be used to report how oxygenation levels change in response to particular, targeted therapies. For example, PAI has been used to monitor intra-tumoral oxygen saturation changes caused by release of catalase and 5-ALA co-loaded nanoparticles 2 levels Label free imaging can be used to monitor changes in tumor hypoxia during a course of treatment. While label free imaging is a superior method to ionizing methods, the amount of information provided by label-free imaging is still limited. In an animal model study where liver metastasis was induced via adenocarcinoma cells, label free imaging confirmed the decrease in overall sOin vivo bioluminescence imaging however, not for tumor hypoxia. Dyes are typically porphyrin and cyanide based hypocrella bambusae prepared with carbon dots Hypocrella bambusae can generate oxygen and has a high red light emission maximum. When combined with carbon dots, the complex is referred to as HBCDs. PAI was used to determine the efficacy of this agents' accumulation in the tumor. Upon irradiation at 635 nm, PA images showed clear tumor microstructure, demonstrating this agent may be an effective tool in photoacoustic-guided tumor therapy The employment of exogenous contrast agents can mitigate some of the shortcomings of label free imaging, as well as amplify detection accuracy The other category of exogenous contrast agents is inorganic 2 extraction. In the presence of oxygen, the dye is activated and turned blue, thus eliciting 'oxygen sensitive' properties. MB has been shown to adequately identify hypoxic regions in pancreatic tumor bearing mice 2 at 650 nm (hypoxic areas) and 810 nm (well oxygenated areas).To estimate physiological changes, organic dye methylene blue (MB) has been implemented due to its oxygen sensitivity 2) have been a popular exogenous contrast agent 2O and O2 and re-oxygenate the area. When testing the efficacy of this contrast agent, PAI was employed and showed poorly oxygenated areas were reoxygenated . Results are shown in Fig. in vitro post administration.Triphenylamine-benzo thiadiazole diethylamino oxide (TBTO) is another dye which is designed to turn into TBT (triphenylamine-benzo thiadiazole diethylamine) due to the over-produced reductases in the hypoxic tumor site 2 is released, resulting in a peak absorption shift to 710nm from 575nm in NR-Azo. Furthermore, this dye has shown to highlight hypoxic regions in vitro on mice implanted with hepatocarcinoma whereas, control mice showed no expression There are other dyes which have similar properties to the aforementioned dyes, however, they undergo activation by different mechanisms. This can render different results as the mechanism of activation depend on different qualities seen in hypoxic environments. Dyes have been developed to take advantage of proteomic features of the tumor microenvironment. For example, NR-Azo is a dye which relies on the upregulation of proteins in tumor hypoxia 2, utilizes a nitrobenzene moiety. This was to take advantage of the overexpression of nitroreductases in the hypoxic environment when 4-nitrobenzene moiety in this agent in vivo xenograft breast cancer model displayed the degree of tumor hypoxia, and also adequately determined the heterogeneity of the hypoxia within the tumor  in vivo.Furthermore, cyanide-based dyes can also render a detailed view of hypoxia in tumors vβ3 is upregulated vβ3, and generates a PA signal at 800 nm. In a study utilizing metastasized hepatocarcinoma cells in mice vβ3 in brain tumor vβ3 targeted gold nanobeacon (αvβ3-GNB), an inorganic contrast agent, was also tested at 740 nm to 810 nm in blood and the authors reported a 10-fold higher PA contrast was observed as compared to the endogenous biomarkers in Organic contrast agents can also take advantage of receptors that are upregulated in tumor hypoxia. In the presence of tumor hypoxia, the integrin αin vivo study designed macrophage-loaded anionic gold nanoparticles (Anionic-AuNRs@RAW) to image tumor hypoxia in a breast cancer animal model With regards to investigating tumor hypoxia based on molecular level alterations using exogenous contrast agents, gold nanorods are most commonly used as an inorganic contrast agent for PAI A similar strategy was pursued by modifying gold nanorods to have 2-nitroimidazole derivatives (NI's), thus making AuNR-NIs in vivo tracking Liposomes conjugated with inorganic compounds and other nanoparticles have been shown to be effective contrast agents According to the published research, PAI is an effective tool to investigate tumor hypoxia. Label free, organic, and inorganic contrast agents are all effective, but each has advantages and disadvantages. A comparison among the contrast agents used in PAI, and corresponding tumor types that have been investigated, are summarized in Table The differences in methodologies and study setups make direct comparison among the various methods difficult. Therefore, the format for the discussion is to explain the application of PAI in characterizing hypoxia with endogenous and exogenous contrast agents by measuring physiological parameters, followed by the use of contrast enhanced PAI to co-localize with tumors.2 and MrO2 can be achieved through the use of endogenous chromophores, HbO2 and Hb. According to the method explained in Section 2, at least a pair of wavelengths  are carefully chosen to illuminate the target area and based on the corresponding absorption coefficients and the concentration of endogenous chromophores, sO2 can be quantified. For MrO2 and OEF calculations, an additional wavelength (605nm) was also utilized. Contrary to expectations, there does not appear to be a direct correlation between increases or decreases in OEF or sO2 and MrO2. Importantly, it was found that early-stage cancers may be hyperoxic instead of hypoxic in spite of hypermetabolism, possibly due to local angiogenesis and tumor size Our review has shown that measurement of physiological parameters sO2 and sO2 can be evaluated based on unique absorption spectra of exogenous contrast agents. Methylene blue (organic dye) was utilized at 680 and 810nm to evaluate pO2. For sO2 calculation, usNP-MnO2 and GE11-PDA-Pt@USPIOS, which are inorganic contrast agents, were utilized in the range of 750-850nm, whereas HC/PN/DOX-NP was used at 660nm.pO2 demonstrating absorption peak shifts from 600 to 740nm and 575 to 710nm respectively. However, other organic agents, ICG based dye and Angiostamp800, is sensitive to overexpressed protein integrin αvβ3 in tumor hypoxia, exhibiting high absorption at ~800nm  and HyP-650 (650 to 740nm) exhibit unique absorbance peak shifts fig.  when expsee Fig. .Inorganic contrast agents such as gold nanoparticles can be useful in determining hypoxia as they can be modified to be guided towards key hypoxic regions within a tumor. Gold nanoparticles may be more advantageous to use as they can be used for drug delivery and re-oxygenation of hypoxic regions. AuNRs (both anionic and nitro-reductase based) provide exciting strategies to assess hypoxia. Absorption spectra of various gold nanoparticles are shown in Fig. While exogenous contrast agents can provide PA images with enhanced contrast, there is a variance in efficacy between inorganic and organic contrast agents. In Table Exogenous contrast agents (organic and inorganic) can take advantage of unique features  of the hypoxic microenvironment to determine the heterogeneity of hypoxia within the tumor itself. Furthermore, exogenous contrast agents can potentially have more robust clinical applications than label free imaging. As the heterogeneity of hypoxia is more visible with these agents, clinicians could potentially predict which lesions will metastasize, as hypoxia promotes a metastatic phenotype The main advantage of PA technology is its potential use in the clinic because it is non-invasive, radiation-free and most importantly, has good penetration depth , highly stable, highly specific, easy to administer, accessible, low cost, and biocompatible for rapid regulatory approval. There is no single contrast agent that meets all the ideal characteristics for every application. The choice of contrast agent for PAI depends on the specific imaging target, imaging system, and application requirements. Further research and development in this field is needed to identify and optimize contrast agents for hypoxia.One limitation of PAI is the need for a coupling medium, such as water or ultrasound gel. In some applications of PA, safely depositing sufficient light energy into the tissue to the required depth is not straightforward. Nevertheless, PA imaging is becoming more common in research labs and development of FDA-approved hypoxia-targeted PA contrast agents should open up many clinical applications for both tumor imaging and adjuvant theranostic applications.2 and MrO2. The advantage of the label-free imaging is that the entire process is non-invasive, however, extraction of hypoxia heterogeneity within the tumor region is not feasible. In addition, we found no studies reporting the correlation between label-free hypoxia and metastasis. Unlike label-free PA imaging, several organic and inorganic agents have been utilized to extract subtle hypoxia changes within the tumor microenvironment and changes to physiological parameters (sO2 and pO2). Organic agents are less risky due to fast clearing as compared to inorganic counterparts. Moreover, organic contrast agents allow better understanding of dysregulated enzyme concentration in the microenvironment and the impact on the disease state. Lastly, studies involving the use of inorganic contrast agents have showcased the potential clinical applications of PAI - particularly the oxygen distribution gradient within the tumor. Since contrast agent-based PA imaging exhibited great potential towards understanding tumor hypoxia, further exploration will potentially allow a more accurate depiction of tumor progression towards possible metastasis, metastasis staging, and appropriate treatment plans. Overall, current literature has shown PAI to be a promising modality in research with a great potential in clinical applications which can lead to more effective treatment plans, enhanced drug development and delivery, and improved patient outcomes.Tumor hypoxia is caused by an insufficient blood supply to a growing tumor. Typically, the tumor peripheral region consists of irregular and dense vasculatures as compared to the core. This dysregulated tumor angiogenesis and lack of perfusion towards the tumor core perpetuates hypoxia and triggers the aggressive phenotypes of cancer cells which could suggest that hypoxia is linked to an increased risk of metastases. In addition, several studies have shown that acute hypoxia corresponds to an increased resistance to chemotherapy and radiotherapy. Consequently, this phenomenon contributes to decreased survival rates for cancer patients with severe tumor hypoxia. Therefore, a highly sensitive, yet non-invasive method, such as PAI is required to study the impact of hypoxia in tumor progression and metastasis. Tumor hypoxia has been investigated with PAI technology to assess physiologic and proteomic phenomena. Endogenous contrast agents , and several organic and inorganic exogenous contrast agents have been explored to map hypoxic regions using PAI. In label-free imaging, hypoxia is primarily studied based on the relation between hemoglobin concentration, oxygenation, and blood perfusion. We now can see that the information provided by label free PAI is enough to determine local sO


Multiple sclerosis (MS) is an immune-mediated disease that targets the myelin sheath of central nervous system (CNS) neurons leading to axon injury, neuronal death, and neurological progression. Though women are more highly susceptible to developing MS, men that develop this disease exhibit greater cognitive impairment and accumulate disability more rapidly than women. Magnetic resonance imaging and pathology studies have revealed that the greater neurological progression seen in males correlates with chronic immune activation and increased iron accumulation at the rims of chronic white matter lesions as well as more intensive whole brain and grey matter atrophy and axon loss. Studies in humans and in animal models of MS suggest that male aged microglia do not have a higher propensity for inflammation, but may become more re-active at the rim of white matter lesions as a result of the presence of pro-inflammatory T cells, greater astrocyte activation or iron release from oligodendrocytes in the males. There is also evidence that remyelination is more efficient in aged female than aged male rodents and that male neurons are more susceptible to oxidative and nitrosative stress. Both sex chromosome complement and sex hormones contribute to these sex differences in biology. MS mostRelapsing-onset MS has a strong female prevalence, which is thought to be a result of females having more robust T cell-mediated autoimmunity [reviewed in ]. By con22.1There is strong evidence that males are more prone to neurological deterioration than females with MS. Males exhibit greater deficits in finger dexterity as measured using the 9 hole peg test , 13 and Male sex is also a predictive factor for having a shorter time to reaching a disability milestone of expanded disability status scale (EDSS)3 and EDSS6 , 27–29; Individuals with MS also experience cognitive deficits including reductions in verbal and non-verbal memory, information processing speed, attention, and executive functioning –36. A laA number of studies have also assessed differences in functional connectivity and neuronal network efficiency in the brains of men and women with MS using functional magnetic resonance imaging (fMRI) and other MRI-based approaches. For example, Schoonheim et al. (2012) investigated the extent of synchronization of activation of different brain regions in the resting state using fMRI . MS pati2.2+ and CD8+ T cells, activated macrophages and microglia, and show evidence of active demyelination and axonal transection and injury . Inactive lesions can be either chronically-demyelinated or myelinated   . Of thesplaques) . In thisplaques) , 50, sugQuantitative susceptibility mapping (QSM) is an advanced MRI method that is used to examine tissue concentrations of paramagnetic iron, which accumulates in activated microglia at the rim of smoldering lesions. A small study of QSM in living MS patients reported that male sex conferred a 10-fold higher risk of having more than one lesion with an active rim, even after adjusting for age, symptom duration, EDSS, and MS subtype . These fIn addition to classic WM lesions, demyelination occurs in the cortices in MS and these demyelinated plaques are referred to as cortical lesions. Cortical lesions are evident even in the earliest stages of MS, but increase in number as the disease progresses . In thisAs MS progresses, there is an increase in the proportion of chronically-demyelinated lesions at the expense of myelinated lesions . This loThe inflammation and demyelination that occurs in both WM and cortical lesions leads to axon injury and loss, which is considered to be the major factor leading to disability and cognitive deficits in MS , 61. A sAtrophy of the whole brain and GM, which contains the cell bodies of neurons, also occurs in MS and the extent of GM atrophy is a predictor of cognitive and disability progression in this disease . With soOptical coherence tomography is a technique that has been used to measure the thickness of the retinal nerve fiber layer. The retinal nerve fiber layer contains the axons of retinal ganglion neurons that continue through the optic nerve, which is targeted by the autoimmune process in MS. Studies in pediatric MS (<18 years) showed that there is more advanced retinal nerve fiber layer thinning in males compared to females with MS, suggesting that sex differences in neuronal vulnerability may be apparent even in children with this disease . Taken t3in vitro and in vivo  is an immune-based model of MS that is most commonly induced in rodents by immunization with protein components of the myelin sheath and adjuvants such as Complete Freund’s adjuvant (CFA) and pertussis toxin  . Immuniz+ T cells are used to induce the disease γ  CD4 disease –98. For  (IFN-)γ . Gonadec (IFN-)γ . Studies (IFN-)γ , indicatin vitro activation with MOG peptide and interleukin (IL) -12  showed that male TCR transgenic T cells that were polarized to Th1 were more encephalitogenic than female Th1 cells upon passive transfer. In this case, the more severe EAE correlated with a higher frequency of the male MOG-specific TCR Tg Th1 cells acquiring a memory effector phenotype upon (IL) -12 . Another(IL) -12 . These f(IL) -12 . Togethe+ T cells are the major players in EAE pathology and MS initiation, CD8+ T cells feature more prominently in WM and intracortical lesions in MS  and cytotoxic  markers and pro-inflammatory cytokines ) ) , 102. Thr (TNF)) , lesiona+ T cells of healthy females have a greater capacity to expand and are more likely to adopt a T effector phenotype compared to male CD8+ T cells, characterized by high IFN-γ and granzyme B production  (a virus that is not implicated in MS) undergoes shifts from a naïve towards an effector memory or senescent status with aging , expresses higher amounts of modulatory cytokines, and acquires cytolytic function only after prolonged activation  in male compared to female microglia in the cortex . Young female microglia express higher levels of pro-inflammatory genes  compared to male microglia after stimulation with IFN-γ; however, when the microglia are passaged, female microglia lose this ability to upregulate these pro-inflammatory factors, whereas male microglia uniquely upregulate IL-1 mRNA in the absence of IFN stimulation  in microglia were increased with middle- and old-age in female mice. This increase occurred, but was less striking, in the males . CompariIt has been difficult in EAE studies to interpret whether there are sex differences in microglia phenotype, since T cells infiltrate the CNS to a greater extent in the female than male mouse [reviewed in ]. NonethIn conclusion, the literature suggests that female microglia are more reactive to IFNs and become pro-inflammatory with age, which contrasts with the finding that men with MS exhibit more smoldering microglia activation at rim of WM lesions during disease progression. It is possible, that the increased microglia seen in male lesions is to an increased responsiveness to damage-associated signals such as ATP, the increased presence of inflammatory mediators secreted by lymphocytes in the perivascular spaces, or enhanced iron release from OLs (discussed in Section 9).8Astrocyte activation also accompanies microglia activation in WM lesions in MS and EAE –168. In glia limitans of the blood brain barrier  in the optic nerve -α specifically in astrocytes, but not in neurons, are resistant to the neuroprotective responses to estrogens . FurtherConsistent with hormone signalling being protective in astrocytes, a histopathological study of female and male MS lesions detected an upregulation of sex hormone receptors in MS lesions compared to the surrounding healthy tissue and NAWM and found that this occurred in a sex-dependent manner . FemalesTaken together, these finding suggest that astrocytes produce and respond to steroids and mediate the neuroprotective effects of ovarian hormones in the brain. There is evidence from animal studies that male rodents may be more prone to develop reactive gliosis during injury and neuroinflammation and that MIF-1, a factor that produced by astrocytes in MS lesions associates with disease progression in males with MS.93+) form bound to ferritin and these stores are contained in OLs and to a lesser extent in neurons, microglia and astrocytes (2+) form, which can interact with oxidants produced by microglia and macrophages to generate the highly reactive and neurotoxic hydroxyl molecule  rats noted the same phenotype of increased proliferation of the female OPCs and a greater ability of the male OPCs to differentiate to mOLs in vitro  are gonadectomized and subjected to cuprizone feeding followed by a period of cuprizone withdrawal, it was found that mice that had an XX chromosome complement had significantly higher electrical conductivity in the CC during the remyelinating phase of disease. This greater recovery correlated with higher proliferation of the female OPCs compared to the male OPCs  are multipotent cells that can self-renew, and migrate to injured WM regions in both EAE  and in c11in vitro studies that male neurons are more vulnerable to the pro-inflammatory molecules and ROS and RNS generated during neuroinflammation. For example, studies in EAE that utilized the four core genotype mouse model provided strong evidence for a role for XX chromosome complement in protecting neurons from damage (-Sry/-) were irradiated (which removes the immune system) and then provided with either XX and XY--Sry/- bone marrow, thereby generating mice that had ovaries and equivalent hormone levels but had either an XX or XY--Sry/- genotype in the radioresistant  or radiosensitive (immune system) compartment. When the radiation bone marrow chimeras were induced to have EAE, XY--Sry/- gonadal female recipients developed more severe clinical symptoms than XX gonadal females regardless of chromosomal sex in the immune system  in the mitochondria  and β-N-oxalyl amino-L-alanine (L-BOAA). For example, treatment of mice with MPTP results in a loss of dopaminergic neurons in the substantia nigra only in male mice, which correlates with lower levels of glutathione mRNA in the male brain; females become sensitized to the neurotoxin when treated with an ER-α antagonist . Similaroduction . In addioduction . Femalesoduction .in vitro treatment of neurons with homocysteine contributes to increased mitochondrial dysfunction and oxidative stress, and stimulates apoptotic and excitotoxic pathways in these cells  [reviewed in ]. Studiese cells . Accordise cells , 225. Onse cells . AnotherTaken together, studies in rodent models suggest that XY neurons are more vulnerable to inflammatory and oxidative stresses compared to XX neurons due to sex differences in X chromosome methylation and effects of ovarian hormones in promoting mitochondrial respiration and antioxidant defense mechanisms. Further homocysteine is a neurotoxic factor that circulates at higher levels in males. If these findings translate to MS, it would give female neurons a survival advantage in the pro-inflammatory environment of the MS lesion.12Gut dysbiosis, which is defined by a reduced diversity or altered composition of bacterial species in the gut, has been reported to occur in MS [reviewed in ]. BesideStreptococcus and S. parasanguinis in SP-MS compared to RR-MS (Streptococcus that can produce hydrogen peroxide. Unfortunately, this study was small and the authors did not disaggregate their data according to sex.Though a large majority of studies of the microbiota in MS have focused on RR-MS, several studies have reported on microbiota changes in progressive MS –239. Altto RR-MS . Metagento RR-MS . The autBacteroides, Enterobacteriaceae and Clostridium g24 FCEY (Clostridium species associated with higher EDSS and fatigue scores in the patients, suggesting that these bacteria could be a driver of disease progression (A study of the CLIMB cohort in Boston reported finding 16 species of bacteria that were differentially abundant between RR-MS and SP-MS patients including g24 FCEY . They algression . Though Akkermansia, and its family, Verrucomicrobiaceae, and Bacteroides spp. are more abundant in healthy female mice (Akkermansia is increased in progressive MS and EAE (Bacteroides spp. compared with mice with chronic-progressive EAE (Sex differences in gut microbiota have been described for both healthy humans and rodents [reviewed in ]. Akkermale mice , 242 andale mice , 244 com and EAE , 245, it and EAE  and in t and EAE . The gutsive EAE  and negasive EAE , are morsive EAE . If thissive EAE –250 (alsLachnospiraceae and a decrease of Erysipelotrichaceae in gut microbiota (Erysipelotrichaceae correlates with brain atrophy in progressive MS, and Lachnospiraceae negatively correlates with fatigue, anxiety, and depression (Akkermansia abundance is reduced when female mice are treated with androgens (Importantly, sex hormones can drive sex differences in gut microbiota  and vicecrobiota . These bpression . Akkermandrogens , and casndrogens .Altogether, sex differences in gut bacteria have been described for healthy mice and humans, and the bidirectional relationship described between sex hormones and gut microbiota suggests that microbiota changes have a potential to drive sex differences in both peripheral and CNS populations that are important to MS progression.13There is more neurodegeneration in males than in females with MS that explains the more rapid physical and cognitive decline in this sex. There are a number of biological mechanisms that may contribute to this more severe neurodegeneration in men (The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.NA-S and SD both contributed to the research and writing of this manuscript. All authors contributed to the article and approved the submitted version.


Imatinib is mainly metabolised by CYP3A4 and CYP2C8 and is extensively bound to α-acid glycoprotein (AAG). A physiologically based pharmacokinetic (PBPK) model for imatinib describing the CYP3A4-mediated autoinhibition during multiple dosing in gastrointestinal stromal tumor patients with normal renal function was previously reported. After performing additional verification, the PBPK model was applied to predict the exposure of imatinib after multiple dosing in cancer patients with varying degrees of renal impairment. In agreement with the clinical data, there was a positive correlation between AAG levels and imatinib exposure. A notable finding was that for recovery of the observed data in cancer patients with moderate RI (CrCL 20 to 39 mL/min), reductions of hepatic CYP3A4 and CYP2C8 abundances, which reflect the effects of RI, had to be included in the simulations. This was not the case for mild RI (CrCL 40 to 50 mL/min). The results support the finding of the clinical study, which demonstrated that both AAG levels and the degree of renal impairment are key components that contribute to the interpatient variability associated with imatinib exposure. As indicated in the 2020 FDA draft RI guidance, PBPK modelling could be used to support an expanded inclusion of patients with RI in clinical studies. The US Food and Drug Administration (FDA) 2010 draft guidance document , this reThe main objective of conducting an RI study is to inform a dose recommendation for the specific population on the drug label. If RI is expected to significantly alter the PK of the drug or metabolite, i.e., renal excretion is the main route of elimination for the drug or metabolite, a clinical PK study in patients with RI is recommended. A reduced clinical study design can be applied initially in non-dialyzed end-stage renal disease (ESRD) or patients with severe RI for drugs mainly cleared by nonrenal routes. Depending on the results, further investigation may be required in other RI classes. The absorption, distribution, metabolism and excretion (ADME) of drugs can change because of accumulated uremic toxins which affect specific processes such as hepatic metabolism, transporter-mediated uptake and efflux, and protein binding ,6. The pTypically, a single-dose RI study is conducted if the drug in development displays linear kinetics, and the same dose is given in both the RI and control groups. Although a multiple-dose study is more appropriate for drugs with non-linear or time-dependent PK, it is recognised that careful consideration must be given to patient groups with a higher prevalence of RI such as those with cancer. Despite this, neither the current FDA guidance 2010) nor the updated guidance (2020) provide clear strategic or decision pathways for RI studies in oncology patients 0 nor the. AccordiHigh-fidelity PBPK models with a sufficient mechanistic resolution of in vivo human clearance routes , are primed for assessing the impact of RI on drug PK in development . Indeed,Key changes in physiological parameters reflecting the effects of RI include reductions of GFR (elevated serum creatinine), inhibition of CYP enzyme or transporter activities (circulating uremic toxins) and a decrease in albumin and an increase in α-acid-glycoprotein (AAG) levels. In cases where a drug binds mainly to albumin in plasma, the increased unbound fraction often reported for patients with renal dysfunction can attenuate the effects of the reduction of CYP activity on total drug exposures; although higher concentrations of the drug are available for uptake into the liver, less metabolism may occur due to the inhibited CYP activity. While AAG represents a relatively small portion (~1–3%) of the total plasma proteins, compared to ~60% composition of albumin, it can still play a significant role in drug binding and PK . Since AImatinib, an anticancer drug and more specifically a tyrosine kinase inhibitor, was approved by the FDA for the treatment of chronic myelogenous leukemia (CML) and gastrointestinal stromal tumors (GIST). As a consequence of its high binding affinity to AAG and its ~55 fold weaker binding affinity to albumin, a non-linear relationship exists between the free fraction of imatinib and total plasma concentrations . Elevatehttp://getdata-graph-digitizer.com, accessed on 24 April 2023). Unless specifically stated, default population files were used in simulations.The Simcyp population-based PBPK Simulator  was used to generate plasma concentration-time profiles of imatinib in healthy subjects and cancer patients with varying degrees of renal impairment. The default SV-ketoconazole file was used in simulations of the drug interaction with imatinib. Clinical study data from the literature were digitized with GetData Graph Digitizer  aged 35–59 years and weighing 64–103 kg were recruited into a drug interaction study between imatinib and ketoconazole . SubjectData from 49 GIST patients  aged 25 to 88 years with acceptable renal function (CrCL ≥ 60 mL/min), receiving imatinib at daily oral doses ranging from 200 to 800 mg were collected over a period of 2 years . Most blSixty cancer patients  aged 16 to 84 years with advanced malignancies and varying degrees of renal dysfunction were recruited into a clinical study to determine the safety, dose-limiting toxicities, MTD, and pharmacokinetics of imatinib and to develop dosing guidelines for imatinib in such patients . FourteeDerivation of a virtual North European Caucasian population  has been described in detail previously . The SimIn the cancer study , patientpop) to calibrate the fu (fupop) from a healthy population value (fu) to that of a disease population, according to Equation (1) [A PBPK model for imatinib published previously was used for the simulations . Drug-retion (1) .(1)fu=1It should be noted that this equation only accounts for changes in fu due to changes in AAG levels and does not consider changes in the binding affinity of a drug that may occur due to accumulated uremic toxins . FurtherThe demographic (including age and gender) characteristics and dose regimen used in the simulations were matched to the clinical studies and the number of virtual subjects was based on 10 trials of the number of subjects used in the corresponding clinical study. In order to verify the contribution of CYP3A4 to the base model (prior to auto-inhibition), we assessed the DDI with ketoconazole in healthy subjects. It should be noted that this had not been done or reported previously for the initial model development . Ten virThereafter, we ran simulations of GIST patients with normal renal function where observed plasma concentration data were available for verification . The modIt should be noted that the combination of the elevated AAG levels with the baseline value fu of 0.05  led to a fu that was significantly lower than the measured value in GIST patients (0.062). Significant interlaboratory variability has been reported previously for measurements of the unbound fraction of imatinib . For calFinally, simulations were run in cancer patients  aged 16 to 84 years with varying degrees of renal dysfunction. Simulations were not performed for severe RI due to the small number of patients recruited into the clinical study (n = 2). Ten virtual trials of 14 cancer patients  receiving daily oral doses of 629 mg for 15 days, 22 cancer patients  receiving daily oral doses of 645 mg for 15 days and 22 cancer patients  receiving daily oral doses of 418 mg were generated and the simulated and observed PK of imatinib [max and AUC ratios  are shown in C ratios , thus coth to 95th percentile of the PBPK model predictions), as shown in After multiple oral doses of 400 mg imatinib in GIST patients, accumulation of the drug occurred; the simulations indicated that only 20% of the assigned CYP3A4-mediated metabolism (40%) remained at a steady state due to autoinhibition of this pathway . DespiteUsing the AAG levels measured clinically for each group, predicted fu values were 0.062, 0.042 and 0.042 for cancer patients with normal renal function, mild renal dysfunction and moderate renal dysfunction, respectively. Corresponding observed values were 0.062, 0.061 and 0.047. Predicted plasma total exposures of imatinib were within 1.25 fold of observed data in cancer patients with normal renal function and mild RI after multiple oral doses of imatinib . AlthougConsistent with the observed data, there was a positive correlation between AAG concentrations and total dose-normalised imatinib AUC on day 15 and an inverse correlation between AAG concentrations and total imatinib CL/F . The simA PBPK model for imatinib describing the CYP3A4-mediated autoinhibition during multiple dosing in GIST patients with normal renal function was reported previously . After aFor drugs of intermediate to low hepatic extraction with a high degree of protein binding such as imatinib, a change in plasma AAG levels or AAG binding affinity can lead to altered total plasma concentrations, with free drug concentrations remaining largely unchanged. However, an increased free fraction can modify the apparent total concentration-effect relationship, confounding the interpretation of therapeutic drug monitoring data which are based on total plasma concentrations . Thus, fDuring drug development, typically, initial clinical studies only include patients with normal or only mildly impaired kidney function, as chemotherapeutic agents used to treat cancer generally have narrow therapeutic indices, along with potentially serious adverse toxicities. When extrapolating to doses for patients with moderate to severe RI, in addition to accounting for physiological changes relating to the cancer itself (captured by the Sim-Cancer population), changes in CYP3A4 and potentially other CYP enzymes may need to be considered. During the development of the Sim-Cancer population, changes in CYP abundance were assessed ( and refeA recent landscape analysis of anticancer agents approved from 2015 to 2019 was conducted to evaluate the inclusion of study participants with CKD and GFR assessment methods used during drug development and subsequent translation to kidney-related safety and dosing data in product labelling . Of the In this study, we have extended the application of a PBPK modelling approach to predict drug PK in patients with advanced malignancies and varying degrees of renal dysfunction and to elucidate the impact of comorbidities in a cancer population, using imatinib as an illustrative example.


In vitro gametogenesis (IVG) has been a topic of great interest in recent years not only because it allows for further exploration of mechanisms of germ cell development, but also because of its prospect for innovative medical applications especially for the treatment of infertility. Elucidation of the mechanisms underlying gamete development in vivo has inspired scientists to attempt to recapitulate the entire process of gametogenesis in vitro. While earlier studies have established IVG methods largely using pluripotent stem cells of embryonic origin, the scarcity of sources for these cells and the ethical issues involved in their use are serious limitations to the progress of IVG research especially in humans. However, with the emergence of induced pluripotent stem cells (iPSCs) due to the revolutionary discovery of dedifferentiation and reprogramming factors, IVG research has progressed remarkably in the last decade. This paper extensively reviews developments in IVG using iPSCs. First, the paper presents key concepts from groundwork studies on IVG including earlier researches demonstrating that IVG methods using embryonic stem cells (ESCs) also apply when using iPSCs. Techniques for the derivation of iPSCs are briefly discussed, highlighting the importance of generating transgene-free iPSCs with a high capacity for germline transmission to improve efficacy when used for IVG. The main part of the paper discusses recent advances in IVG research using iPSCs in various stages of gametogenesis. In addition, current clinical applications of IVG are presented, and potential future applications are discussed. Although IVG is still faced with many challenges in terms of technical issues, as well as efficacy and safety, novel IVG methodologies are emerging, and IVG using iPSCs may usher in the next era of reproductive medicine sooner than expected. This raises both ethical and social concerns and calls for the scientific community to cautiously develop IVG technology to ensure it is not only efficacious but also safe and adheres to social and ethical norms. In vivo gametogenesis is a complex process, and an extensive understanding of its molecular mechanisms is crucial for understanding reproductive health and associated diseases, such as infertility. Unfortunately, studies on germ cells raise several ethical issues Aoi . There iEarlier studies on IVG involved the use of pluripotent stem cells (PSCs) of embryonic origin. However, this approach does not obviate the problem of scarcity and the ethical issue of using embryonic cells. A novel approach that overcomes these concerns is the use of induced pluripotent stem cells (iPSCs) rather than PSCs of embryonic origin , which are in the primed state with a biased differentiation potential and are thus recalcitrant to germ cell differentiation and chimaera formation . Primed mEpiSCs, on the other hand, express genes encoding primed transcription factors, such as Oct6, Otx2, Sox2, Bex1 and Tead2, as well as the core pluripotency genes  using embryoid bodies that spontaneously differentiated under undefined conditions  had genetic and epigenetic signatures consistent with those of in vivo PGCs. Upon transplantation into neonatal testes, these epiPGCs developed into mature spermatozoa capable of fertilizing oocytes and producing healthy offspring  formation using random differentiation strategies resulted in very inefficient and inconsistent PGC specification (only approximately 0.5–3.6% per EB), a direct induction method was deemed necessary for PGC specification in vitro -positive ESCs from E3.5b mouse blastocysts and maintained these cells in the ground state using 2i + LIF feeder-free culture. These ESCs were then stimulated with ActA, bFGF, and 1% KSR for 2 days to produce what they called epiblast-like cells (EpiLCs). Based on an analysis of the expression of key genes in day 2 EpiLCs, they demonstrated that these cells had properties that corresponded to those of E5.75 pre-gastrulating epiblasts, thus suggesting that they were germline competent precursors capable of differentiation into PGCLCs  transgenes and expressed NG in the ground state, the three lines exhibited different SSEA1 and Integrinβ-3 FACS sorting patterns at day 6. Only one iPSC line (20D17) was very similar to the ESC lines. Likewise, only this 20D17 line exhibited proper spermatogenesis after being transplanted into W/Wv mouse testes. These findings show that despite iPSCs exhibiting different induction patterns depending on the iPSC line, they can nevertheless be induced into PGCLCs that function properly  has presented a promising alternative to cells of embryonic origin, making it possible to elucidate aspects of germ cell biology that have been unexplored due to material limitations and ethical concerns . Although OSKM have been the four most popular reprogramming factors, iPSCs have also been generated using the expression of Oct-4, Sox2, Nanog, and Lin28  together with BLIMP1 was noted. In association with SOX17, BLIMP1 not only suppresses somatic differentiation by itself but also promotes germ cell specification , as an intermediary step from hiPSCs to hPGLCs. The hiPSCs were first activated using a GSK3 inhibitor (CHIR99021) and activin A to induce iMeLCs that expressed pluripotency genes as well as genes for mesoderm development . These cells were then placed in suspension culture using BMP4, EGF, SCF and LIF to convert the iMeLCs into BVSC-positive hPGCLCs that expressed Blimp1, Oct4, Tfap2c, Sox17 and Nanog, which are all early germ cell markers in humans. Gene Ontology analysis after RNA-seq showed that Sox17 and Blimp1 are crucial regulators of hPGCLC specification  that then produced embryos in a gestational mother . These SSCs can self-renew and produce haploid gametes and spermatocytes through association with Sertoli cells -prospermatogonia on the 120th day in the ALI system. Single-cell RNA-seq analysis of gene expression patterns showed that these in vitro-derived prospermatogonial cells were equivalent to their corresponding in vivo counterparts. However, the functionality of these cells was not assessed  but not the genes for meiotic recombination. The transcriptomic profiles of these oogonia-like cells from hiPSCs were akin to those of W7 oogonia and W9 gonocytes of embryos in humans  and H19 loci  + BVSC system. They showed that day 4 mPGCLCs in 5-day culture with FR10Cs5  subsequently combined with E12.5 testicular somatic cells to form rTestes (reconstituted testes), which had the best results in producing SLCs according to the ratio and number of VR-positive cells in the ALI system for at least seven days. Once these SLCs are transplanted into testes and the testicular transplants are cultured in vitro, they differentiate into mature functional sperm -positive primary oocytes and Foxl2+ granulosa cells similar to those in primary follicles  from hiPSCs/hESCs   . Other promising therapeutic applications for IVG using iPSCs include but are not limited to 1) fertility preservation for prepubertal children needing to undergo gonadotoxic chemotherapy (Pampanini et al. The challenges that face IVG using iPSCs are largely issues of efficiency, safety and ethicality (Saitou and Hayashi Another limitation that IVG using iPSCs must overcome is safety (Makar and Sasaki -derived gametes are still remote, with the rate at which science and medicine are progressing, some clinical applications may arrive sooner than expected (Cohen et al. There are also ethical and social concerns that must be considered when using iPSC-derived in vitro gametes. Although clinical therapeutics using in vitroFrom its proof-of-concept studies, the science of IVG has advanced at a very rapid pace. The ground-breaking discovery of iPSCs and their use for the creation of these gametes have contributed enormously to this progress. Several IVG techniques and models using iPSCs have been developed in both mice and humans. Despite this rapid advancement, the efficient and reliable recapitulation of gametogenesis in its entirety in vitro still eludes us. Although oogenesis has been achieved entirely in vitro in mice, the efficiency of the process and the rates of successful term births of the resulting pups remain low. Emerging techniques such as the use of synthetic embryos and the direct induction of germ cells using transcription factors may improve the efficiency of IVG using iPSCs and could facilitate the complete in vitro reconstitution of mature human gametes. Although some scientists believe that the translational applications of IVG are limited to better elucidating germ cell biology, it is already being used to model certain conditions that cause infertility in order to better understand the underlying pathogenetic mechanisms on a cellular level, with the aim of discovering therapeutic options for diseases that are currently untreatable. IVG holds great potential for reproductive medicine and could usher in the next era of reproduction and regeneration. Nevertheless, as with any breakthrough technology, along with this enormous therapeutic potential comes great risk and many ethical responsibilities. The scientific community should continue to cautiously advance technology related to IVG and iPSCs and pay special attention to improving the efficiency and safety of the processes, while also ensuring that they are ethically sound.


We present retrospective data from our clinical research efforts of the past several years alongside a review of past and current clinical and preclinical data independently by several investigators supporting our clinical evidence for the importance of inflammation in inherited retinal degenerations (IRDs). We show how inflammation is a complicating factor in IRDs but, if recognized and managed, also a great opportunity to mitigate disease severity immediately, improve patient prognosis and quality of life, extend the treatment windows for gene-specific and agnostic therapeutic approaches, mitigate the impact of inflammatory complications on the accurate estimate of vision changes in IRD natural history studies, improve the chances of safer outcomes following cataract surgery, and potentially reduce the likelihood of inflammatory adverse events and augment the efficacy of viral vector-based treatment approaches to IRDs. Manuscript contribution to the field. Inflammation has been suspected to be at play in IRDs since the beginning of the 1900s and became a research focus through the early 1990s but was then largely abandoned in favor of genetic-focused research. Thanks to regained cognizance, better research tools, and a more holistic approach to IRDs, the recent reappraisal of the role of inflammation in IRDs has brought back to the surface its importance. A potential confounder in natural history studies and a limiting factor in clinical trials if not accounted for, inflammation can be managed and often offers an opportunity for immediately improved prognosis and outcomes for IRD patients. We present our retrospective clinical evidence for connections with a measurable secondary autoimmune component that can develop in IRDs and contribute to vision loss but is at least in part treatable. We also present ample lines of evidence from the literature corroborating our clinical observations at the preclinical level. The most apparent clinical change related to ongoing inflammation at the tissue level is the presence of cystoid macular edema (CME), a well-known and common complication of IRDs. In addition to CME, virtually every IRD specialist has seen IRD patients also present with retinal exudates, perivascular sheathings, vascular staining, and/or leakage of the far peripheral vessels or at the vascular arcades on intravenous fluorescein angiography (IVFA). It has been our experience that far less appreciated appear to be signs of late leakage on IVFA at or around the optic nerve head and swelling of the retinal nerve fiber layer (RNFL) on optical coherence tomography (OCT)—but it has been our experience that they are also present. The less frequent appreciation of these features is in part since inflammatory disc changes in IRDs tend to be best appreciated in the very late IVFA frames (>6 min), and that glaucoma specialists or neuro-ophthalmologists routinely ask for RNFL OCTs, but not by IRD—or more in general, retinal—specialists.Inherited retinal degenerations (IRDs) comprise a genetically and clinically heterogeneous group of conditions due to mutations in over 300 distinct genes characterized by the common leitmotif causing the progressive degeneration of photoreceptors and vision loss . The mecThe presence of these inflammatory findings in IRDs led researchers in the field to investigate, in the late 1980s, the immune system reactivity against retinal antigens in patients with RP . In earlThe early investigations into the role of the immune system in RP pathogenesis and progression led us to investigate the therapeutic potential of an IMT agent available in the 1990s, thymopentin . AdminisOver 3 years have gone by since the time when the role of the immune system in IRDs was being first investigated, and this hypothesis was set aside as the focus of IRD researchers rapidly shifted towards the discovery of new genes and the characterization of genotype-phenotype correlations. More recently, though, there has been a progressive reappraisal of the role of inflammation and activation of the immune system in IRDs . SeveralSome investigators have questioned the potential pathogenicity of AR-AAbs, and the need for standardized testing methods has been emphasized . In prioDespite all these lines of evidence, detection of the very same AR- or AON-AAbs is not presently considered equally relevant in IRDs, and is most often dismissed as a mere after-the-fact secondary marker of prior degeneration. However, our clinical experience with many IRD patients has been quite different. IRD patients exhibiting inflammatory complications virtually invariably present also with AR- and/or AON-AAb patterns that correlate quite well with clinical and functional findings, especially at the retinal immunohistochemistry (rIHC) testing level, and respond favorably to the treatment of these complications. This is in line with a pathogenic view of these AAbs also in IRDs . Thus, wWe conducted a retrospective analysis, approved by the Duke Institutional Review Board, of subjects seen at the Duke Center for Retinal Degenerations and Ophthalmic Genetic Diseases who had been referred for a possible diagnosis of IRD but also exhibited inflammatory signs and symptoms, thus, underwent AAb and rIHC testing between 2016 and the summer of 2022 according to published methods . The stuPatients meeting clinical or imaging criteria that we defined as suggestive or outright indicative of inflammatory complications during their examination  underwenvice versa. In the vast majority of cases, AAb-positive patients were also positive for rIHC testing which allowed us to establish such cellular level correlates with precision. For example, a patient presenting with inflammatory optic nerve findings (disc swelling on SD-OCT and leakage on IVFA) may well present without AON-AAbs but exhibit AR-AAbs and RNFL and/or RGC staining on rIHC, whereas one with mild if any disc changes but exhibiting PVEP delays suggestive of an inflammatory optic nerve component may present exclusively with AON-AAbs and no AR-AAbs and no RNFL and/or RGC staining on rIHC. Also, patients presenting with more complex pictures such as electronegative ffERG due to selective or prevailing b-wave compromise would most often exhibit retinal bipolar cell (BC)/inner nuclear layer (INL) and/or outer plexiform layer (OPL) staining and/or AR-AAbs directed against known BC/INL- and/or OPL-expressed antigens—or even against arrestin, a protein of the visual cycle that, when compromised genetically like in Oguchi disease  suggestive of a likely IRD, and/or a diagnosis of an RP or macular dystrophy—e.g., Stargardt disease—in childhood also underwent CLIA-certified molecular genetic testing to confirm the presumed or suspected diagnosis of an IRD. Whenever possible, samples from other affected members  or from the presumed healthy carrier parents in suspected recessive disease were also obtained and tested as part of the diagnostic process to establish the phase of any detected gene mutations and confirm the pathogenicity of detected mutations. All patients underwent as a minimum IRD-focused broad CLIA-certified next-generation sequencing (NGS) testing. The most commonly used labs for diagnostic molecular genetic testing included GeneDx  and the Molecular Vision Lab . Initially negative or inconclusive results generally led also to escalation to deletion/duplication analyses and/or whole exome sequencing (WES) testing when the suspicion of an IRD was especially high. Further details about our approach to molecular genetic testing and diagnostic management of our IRD patients have been recently published .EYS and USH2A mutations—these are subsets of genetically well defined patients in which we initially empirically noted an unusually high frequency of inflammatory findings and that have been presented but not published before  analyses of our findings in genetic subgroups of patients harboring d before . These in = 261) had either primary AIR or (more commonly) AINR/ARRON or a paraneoplastic form thereof (CAR/CARON). The latter diagnoses were established based on age and modality of presentation . AON-AAbs were found in 8 out of 9 tested patients. AAbs against both tissues were seen in 6 patients, and rIHC showed positive staining in 9 of 12 cases, labeling predominantly photoreceptors (8/12) and less frequently RGCs and the RNFL. The specifics of the observed autoreactivities and related correlations are illustrated in In 2021(76), we retrospectively identified 20 subjects positive for biallelic disease-causing Altogether, 70% (14/20) of patients exhibited subclinical signs of inflammation and, in 12 of them, they were associated with an autoimmune component that correlated closely with imaging and functional findings. These patients received intravitreal and/or sub-Tenon steroid injections, with both subjective and measurable increases in vision , associated with improved SD-OCT and IVFA characteristics in most.USH2A gene mutations, all of whom had a complete eye exam, including visual acuity (VA), visual fields (VFs), ffERG, macular (n = 75) and, in many, optic nerve (n = 40) SD-OCT, IVFAs in about 45% of the subjects (n = 31), and CLIA-certified testing in 50% (n = 35) for circulating AR-AAbsand/or AON-AAbs by antigen-speficic immunoblot, WB, and rIHC.In 2022 , we retrOf the 62 tested eyes tested with IVFA, 38 eyes had leakage of optic nerve head, vascular arcades, macula, or a combination thereof. The RNFL was thickened on SD-OCT, most often sectorally, in 49 of 80 eyes, and correlated well with IVFA leakage, helping explain disproportionate VA losses compared to foveal SD-OCT findings. CME was seen by SD-OCT in 47 of 150 eyes (31%). AR-AAbswere found in 32 of the 35 tested patients [most often against carbonic anhydrase II (16/35) and enolase (15/35)]. AON-AAbs were found in 28 of 34 tested patients, and rIHC showed positive staining in 28 of 34 cases, labeling predominantly photoreceptors (26/34) and less frequently RGC (11/34) and RNFL (8/34). Altogether, 66.7% (50/75) of patients exhibited subclinical signs of inflammation, which correlated directly with the presence of circulating AAbs in 25 of them. The specifics of the observed autoreactivities and related correlations are illustrated in Also, these patients, after modest to no response to initial trials of topical and/or oral CAIs for CME and in all cases when significant optic nerve inflammatory involvement was present (which would not be expected to respond to CAIs), received intravitreal and/or sub-Tenon steroid injections in doses and frequencies dictated by the severity of the observed inflammatory findings, with or without subsequent addition of IMT regimens based on response to the intra/periocular steroids. Most patients experienced both subjective and measurable increase in vision , associated with improved SD-OCT and IVFA characteristics in virtually all treatment-responsive cases.RHO-linked autosomal dominant (AD) RP will be re-reviewed and illustrated in further detail here, alongside a representative case of EYS-associated ARRP and even one with ABCA4-associated cone-rod dystrophy (CORD). These 3 cases have been specifically chosen to emphasize the very important role of optic nerve inflammatory complications, in IRDs, well beyond CME  and how treatment of these cases can allow patients in glaring cases like these ones to achieve their “true retinal potential”.How much vision can IRD patients recover from ongoing inflammatory complications? The short answer is—it depends on the severity of the complications at the time of evaluation, the timing of the identification of the complication with respect to any more sudden-onset, abrupt vision changes, and the stage of the underlying disease itself. We have previously briefly reported two cases of IRDs who experienced marked visual loss recovery after management of inflammatory complications associated with detectable AR- and AON-AAbs . One witThis 51-yo White female [briefly reported in (80)] presented with a family history of RP consistent with dominant inheritance. BCVA was 20/20–2 in the right eye and 20/30 in the left. However, the patient complained of blurred vision. Fundus examination showed altitudinal RP limited to the inferior retina , arrows,RHO P23H mutation. Because of the VF/ffERG and fundus exam mismatch, disc inflammation was also suspected. PVEPs were obtained (not shown), exhibiting marked delays at all tested spatial frequencies  without amplitude response loss. An IVFA . Typical features of ABCA4-related disease such as peripapillary FAF sparing and hyperautofluorescent flecks peripheral to the atrophic spots were noticeable. At this stage, the patient was not subjectively noticing any VF loss and VF was normal to confrontation.A 29-year-old White male with a history of “Stargardt” since childhood presented with a best corrected visual acuity (BCVA) 20/200 with severe loss of ffERG and a widespread phenotype characterized by disseminated atrophic spots, compatible with an advanced stage CORD . The patThis initial presentation was followed by a sudden, precipitous bilateral BCVA to 20/400 and severe subjective VF loss, which was confirmed by SKP testing . A Full-EYS, USH2A, MERTK, CRB1, BBS1, NR2E3, ABCA4, RHO, RP1, KLHL7, and the PRPF family of genes were among the others more commonly associated with AAb-associated inflammatory manifestations—and among the latter ones, the PRPF31 gene was more commonly so than the PRPF8 one. It is not presently known if more immunogenic phenomena associated with certain genotypes may be behind the clustering of AAb autoreactivities within the aforementioned genes. Based on the evidence associated with MERTK gene mutations (reviewed in further detail below), this would be expected to be the case in conjunction with this one gene and may also be the case for the others. This aspect deserves further investigation at the preclinical level. For BBS1, the association we found is also quite interesting in light of very recent evidence pointing to this gene, and potentially more in general the BBSome, in being involved in immune synapse assembly by promoting the centrosome polarization to the antigen-presenting cells and in the regulation of selective functions of T cells that predispone BBS patients to other autoimmune and inflammatory diseases . Contrary to a common belief that virtually any IRD patient tested will exhibit autoreactivities, we had 9 subjects who, despite being suspected of having secondary autoimmune complications due to the presence of visible or strongly suspected inflammatory complications, tested negative for any type of AAb and exhibited no staining upon rIHC. Thus, despite the sample being upfront more likely to exhibit autoreactivies by virtue of the criteria used to make it medically appropriate to order AAb and rIHC testing, not even everyone in this enriched sample always showed autoreactivity. Our findings show that secondary autoimmune complications in IRDs are indeed common and appear to be more associated with certain genotypes than others. In our retrospective case series, the diseases .The work of Adamus et al.  on the RMertk-deficient mice and we observed the same type of anti-arrestin and anti-IRBP in the sera of these mice , worldwide approved for medical use, which potently suppresses de novo guanine nucleotide production by reversibly inhibiting inosine monophosphate dehydrogenase (IMPDH). Yang et al. have discovered that intraperitoneally administered MMF suppresses retinal cyclic guanosine monophosphate (cGMP)-dependent photoreceptor toxicity in both the rd1 and rd10 ARRP mouse models, both caused by PDE6B mutations  microglial morphological and migration patterns, and far less photoreceptor layer infiltration by microglial cells. While microglial activation may be important to remove debris resulting from inflammatory events, they also trigger a cascade of inflammatory events, as it has been shown in the Mertk murine models discussed above that are connected both directly and indirectly to their migration to non-physiological retinal locations. Thus, notwithstanding the noted cGMP-related effects that make MMF and derivatives thereof attractive as an outright treatment candidate for various forms of RP, a benefit to the inflammatory side of IRDs through its impact on local intraretinal inflammation pathways provides an important validation for its observed clinical benefits in IRD patients with inflammatory, autoimmune-associated complications.As mentioned in the introduction section, there is now evidence that some IMTs may exert outright beneficial effects on IRDs utations . After tvia the inhibition of purine and pyrimidine synthesis, transmethylation reactions, translocation of nuclear factor-κB to the nucleus, signaling via the Janus kinase–signal transducer and activator of transcription (JAK–STAT) pathway and nitric oxide production, as well as the promotion of adenosine release and expression of certain long non-coding RNAs  of MTX were then tested in vitro in the P23H RHO knock-in mouse model for potential therapeutic efficacy at stages when rods were still sufficiently intact to respond to treatment. A regimen of four weekly 25 pmol IVIs started at post-natal day 15 increased misfolded P23H clearance via the lysosomal pathway and autophagy also in vivo, and led to significantly better functional (ffERG), increased outer segment rhodopsin levels and better ONL preservation compared to untreated mice. A single IVI was insufficient to attain these results, and the 25 pmol dosing outperformed 100 pmol, indicating that there is an optimal therapeutic range for MTX IVIs. Since MTX is a widely available drug and worldwide approved for systemic medical use, and is also available as for IVI regimens in humans, Aldeyra Therapeutics has initiated a human clinical trial (NCT05392179) of IVIs of MTX in ADRP patients with P23H RHO mutations and other such mutations in which rhodopsin misfolding and aggregation has already been shown to occur  and external (or outer) limiting membrane (ELM/OLM) or RNFL loss, especially if affecting the papillo-macular bundle (PMB)] and more modest IVFA findings. Most of these patients reported, in their prior history, slow progression of their vision loss over the years until they experienced a sudden acceleration and relatively rapid vision loss that could not be otherwise explained by previous treating physicians. This suggests in these poorly responsive cases that the autoreactivities, at these “burnt-out” stages, truly reflected biomarkers of likely is at the end of the tunnel for IRDs. We sincerely hope that, having shared this experience, the same caution and prudence in preevaluating  IRD patients that we use ahead of cataract surgery will be used by everyone henceforth.A common account reported by IRD patients exhibiting autorectivities was also the exacerbation of vision loss within months of undergoing cataract surgery, performed elsewhere, whereby some patients had subsequently refused to have the fellow eye operated on out of fear of experiencing the same outcome. It has been recently reported that CME, the most frequent post-operative inflammatory complication of cataract surgery  is much more common in RP patients than in the general population . There iMertk-deficient mice (In summary, AAb-associated inflammation in IRDs is not a mere epiphenomenon and, at least in many cases, it is truly impactful yet treatable, in line with the mechanistic framework already proposed by Adamus . Thus, went mice . Ultimat


Chronic obstructive pulmonary disease (COPD) is a progressive disease characterized by airway limitation and changes in airway structure. It has a high global burden of mortality and morbidity. The etiology of COPD is complex, but exposure to tobacco smoke and other inhaled lung oxidants are major risk factors. Both pharmacological and non-pharmacological approaches are used to manage COPD, but there remains an urgent unmet need for drugs that can modify the course of the disease. This review focuses on the role of acetylcholine and other components of the pulmonary cholinergic system in the pathogenesis of COPD, and the inhaled pharmacological agents that target it. In addition to its role as a neurotransmitter, acetylcholine regulates diverse aspects of COPD pathogenesis including bronchoconstriction, airway remodeling, mucus secretion and inflammation. Inhaled antimuscarinic drugs are a key component of therapy for COPD, as monotherapy or in combination with inhaled β2 agonists or corticosteroids. We review the evidence supporting the use of current anticholinergic agents in COPD and preview novel drugs targeting the cholinergic system and agents from other classes in clinical development, such as phosphodiesterase-4 inhibitors and monoclonal antibodies targeting inflammatory mediators. Patients with COPD are at risk of acute intensification of respiratory symptoms (‘exacerbations’) arising from respiratory infections or environmental triggers that may lead to hospitalization  -5), benralizumab (anti-IL-5 receptor), dupilumab (anti-IL4 receptor) and tezepelumab  . Most mADue to pathological similarities between asthma and COPD, mABs approved for severe asthma are undergoing clinical evaluation for COPD . In asthDupilumab is a mAB inhibiting IL-4R, a component of the type 2 receptor complex (IL-4/IL-13) that regulates recruitment of eosinophils as well as airway remodeling in asthma . Two cliIn addition to mABs approved for asthma, other biologics targeting inflammatory pathways have undergone evaluation for COPD. Infliximab, an inhibitor of soluble and membrane-bound tumor necrosis factor-α (TNF-α) has been evaluated in phase 2 studies enrolling patients with mild, moderate and severe COPD, but no clinical benefits were reported ,92. FurtIn this review, we focus on the role of acetylcholine and other components of the pulmonary cholinergic system in the pathogenesis of COPD, especially in the epithelial cell inflammation, mucus plugs, small airway dysfunction and remodeling. We also emphasize the anti-cholinergic agents for treatment of COPD. Currently, COPD is a disease with a high unmet need for therapies that modify the underlying disease processes rather than alleviate symptoms. As the pathogenesis of COPD is further elucidated, new data will assist the development of novel agents targeting both the cholinergic system and other pathways contributing to the disease. A better understanding of disease biomarkers may inform a targeted approach to matching individual COPD patients to the most appropriate selection of therapy.The ambiguous clinical efficacy of mABs for COPD in trials thus far is likely due to a redundancy in pathways responsible for inflammation, as well as the heterogenous populations recruited into large clinical trials. Trials enrolling more narrowly defined populations may be of great value in identifying the precise role of biologics in the future treatment strategies for COPD.