Investigators from Harvard University and UCLA have reported that despite evidence of structural abnormalities in the visual pathway of animal models and children with tuberous sclerosis complex (TSC), visual evoked potentials (VEPs) in 12-month old children with TSC compared to an age-matched control group are not significantly altered. Investigators from Harvard University and UCLA have reported that despite evidence of structural abnormalities in the visual pathway of animal models and children with tuberous sclerosis complex (TSC), visual evoked potentials (VEPs) in 12-month old children with TSC compared to an age-matched control group are not significantly altered. This study is motivated by the high rates of autism spectrum disorder in children with TSC, which the authors posit may be due to delayed visual processing.This multisite, longitudinal case-control study compares 16 infants with TSC  to 18 “typically-developing infants.” Infants were presented a black and white phase-reversing checkerboard during which visual fixation was tracked, and 50-100 trials were recorded. Peak amplitude and latency of the N1, P1, and N2, the primary components of the visual evoked potential, were measured and were found to be not significantly different in a non-parametric analysis. [TSC1 in the retina, both the photoreceptor layer itself and the axon bundle are severely disrupted [TSC2 gene mutation leads to enlarged photoreceptor and support cell units (ommatidia) with failure to prune synaptic connections [TSC2 mice has implicated ephrin-EphA receptor signaling in abnormal development of retinogeniculate connections [Tsc2 activation. When taken in the context with studies of patients with TSC showing that a delay in nonverbal ability, primarily visual processing, was an early biomarker for cognitive impairment [COMMENTARY. This paper seeks to identify an early and easily-tested surrogate for neurodevelopmental impairment in children with TSC, as increased rates of autism in the TSC population suggest the increased prevalence in this population might warrant early therapeutic interventions in the future if possible. In Drosophila lacking isrupted ; similarnections . Furthernections , presumapairment , the autAs measurement of a visual evoked potential (VEP) surveys the visual pathway from retina to primary visual cortex, any aberration in synaptic connectivity along this pathway would be expected to impact latency or amplitude in a population-based fashion. VEPs would also prove useful in a clinical setting, as infants with limited ability to cooperate could be tested at different age intervals. However, despite known MRI abnormalities in the occipital region and concurrent diagnosis and treatment of an epileptic encephalopathy, infants in this study did not manifest any systematic difference in VEPs. Potential explanations for this interesting yet unexpected negative finding include the level of visual processing studied - for example, while a previous study noted specifically a deficit in face processing in individuals with TSC , this hi


Acetazolamide (ACZ) is a sulfonamide derivative that inhibits carbonic anhydrase and is the mainstay for prevention and treatment of acute mountain sickness (AMS). Acute kidney injury (AKI) is not well recognized as a complication of ACZ ingestion, especially when low doses are used for short periods of time. We report a case of a healthy, middle-aged man who developed severe AKI after the ingestion of ACZ for AMS prophylaxis. The patient presented with bilateral flank pain and anuric AKI without radiographic signs of obstructive uropathy. All blood and urine testing to determine the cause of AKI were negative or normal. The patient required 2 sessions of hemodialysis due to worsening metabolic derangements, which included severe anion gap metabolic acidosis and hyperphosphatemia. Renal function returned to baseline after 96 hours of supportive care. The pathogenesis of AKI in our patient was attributed to ACZ-induced sulfonamide crystalluria causing intratubular obstruction and retrograde urine flow, but not intraureteric precipitation or obstructive uropathy. This classic presentation of anuric AKI and renal colic has been previously described with higher doses of ACZ for prolonged periods of time but never with low doses for AMS prophylaxis such as in our patient . Our case highlights the risk of adverse renal outcomes following ACZ ingestion, even in previously healthy individuals, and suggests that increased fluid intake may be advisable for travelers taking ACZ prophylaxis. Additionally, prophylactic and therapeutic agents must be dose-adjusted and interactions reviewed, particularly in the setting of other comorbidity or medications for traveler’s diarrhea or malaria.3Ascent to high altitudes is common for both occupational and leisure activities. When the ascent is >2350 m, there is risk of high-altitude illness: acute mountain sickness (AMS), high-altitude cerebral edema, and high-altitude pulmonary edema. AMS is the most common form of high-altitude illness, and its incidence is about 50% at 2800 m.4 Evidence has shown that low doses of 250 mg per day may be equally effective compared to higher doses.5 We report a case of severe acute kidney injury (AKI), an underrecognized complication of ACZ, in a healthy middle-aged man after the ingestion of ACZ for AMS prophylaxis.Acetazolamide (ACZ) is a sulfonamide derivative that inhibits carbonic anhydrase (CA) and was approved by the Food and Drug Administration in 1953 for its use as a diuretic, anticonvulsant, and antiglaucoma agent. ACZ is rapidly absorbed, achieves a peak concentration at 2 to 4 hours, and is eliminated unchanged in the urine. ACZ is also the mainstay for prevention and treatment of AMS.2 of 30.5 mm Hg, anion gap of 31 mmol/L, potassium of 5.1 mmol/L, and phosphorus of 7.3 mg/dL. The patient received intravenous (IV) isotonic fluids  with minimal response and subsequently required renal replacement therapy  because of persistent anuria with worsening metabolic derangements  250 mg before ascent to Ancash, Peru (4500 m above sea level) and 3 doses of 250 mg while at peak ascent, all at intervals of 12 hours . He had taken similar prophylaxis without incident on previous trips to high altitudes. Twenty-four hours after peak ascent, he developed headache, nausea, bilateral low back pain, and oliguria. He received 1 injection of ketoprofen when he initially sought medical attention in an acute care center within the first hours of symptom onset. However, due to persistent symptoms, he returned to Lima, Peru (1550 m above sea level) to seek further medical attention. On presentation, his physical examination was unremarkable and vital signs were within normal limits. His initial evaluation included a kidney ultrasound that showed normally sized kidneys, uncomplicated right-sided nephrolithiasis without obstruction  and plasma bicarbonate concentration, ultimately lowering CSF pH and stimulating ventilation through central chemoreceptors.11 Additionally, inhibition of CA in red blood cells or vascular endothelial cells may cause carbon dioxide retention and tissue respiratory acidosis12 and thereby stimulate increased ventilation.13 Other therapeutic actions of ACZ include reduction in the number of bedtime arousals (apnea-associated hypoxemia) and improvement in the quality of sleep at high altitude by reducing periodic breathing ,15 an effect that is likely mediated by ACZ inhibition of peripheral chemoreceptors.16 Finally, the inhibition of renal CA induces water and salt losses in the kidney and may balance the state of increased aldosterone at high altitude, an adaptive response to plasma volume depletion and water losses from prolonged hyperventilation.17AMS occurs due to hypoxia-mediated mild cerebral edema and increased intracranial pressure18 in 1956 in a patient with Hodgkin’s disease who received a 4-day course (750 mg per day) of ACZ as a diuretic and was found to have coagulum and sulfonamide crystals in the renal tubules on postmortem analysis. Similarly, autopsy of a patient with nephrotic-range proteinuria who developed thrombocytopenic purpura and renal failure following exposure to ACZ as a diuretic (3000 mg in a period of 9 days) revealed dilated renal tubules containing casts and birefractive crystals, which resembled those obtained by recrystallizing ACZ in an acidic medium.19The first description of renal lesions but not renal failure after treatment with ACZ was made by Glushien and Fisher20 described a case of “sulfonamide crystalluria” in a 54-year-old man exposed to ACZ for glaucoma  who had severe low back pain, hematuria, azotemia, and anuria without evidence of kidney stones but with bilateral ureteral obstruction that required emergent catheterization. In this patient, the urinalysis revealed RBCs but not crystals or casts. The patient received oral bicarbonate therapy and recovered kidney function close to baseline by the time of discharge. In 1975, Howlett21 reported a case of a 69-year-old man who received a 2-week course of ACZ for glaucoma (1000 mg per day) and presented with anuria, azotemia, nausea, and flank pain. Urine microscopy showed many RBCs but not crystals on multiple evaluations. The patient received oral bicarbonate therapy and oral hydration and fully recovered kidney function. In 1978, 2 cases of “acute hemorrhagic anuria” following courses of ACZ  were reported. Both patients presented with back pain, progressed to anuria, and had antegrade urograms that revealed dilated renal pelvices and mucosal swelling or sludge-like material blocking the ureters. There was full recovery of kidney function following the release of the obstruction in both individuals.22In 1958, Yates-Bell23 reported the case of a 76-year-old woman who was exposed to ACZ for glaucoma (425 mg per day for 3 weeks) and presented with anuric AKI without evidence of urologic obstruction or crystalluria. The patient responded to IV hydration and her kidney function returned to normal after 48 hours of supportive care. A similar case was described by Lopez-Menchero et al,24 in which a 58-year-old man who was taking ACZ for serous maculopathy (750 mg per day for 4 days) presented with low back pain and anuric AKI without radiographic evidence of obstruction or thrombosis. Urine microscopy showed RBCs but not casts or crystals. Again, the patient responded to IV hydration with normal saline and bicarbonate and fully recovered kidney function after 36 hours of hospital admission.More recently, Epelde Gonzalo et al25 in a 45-year-old man who was taking ACZ after a trabeculectomy of the left eye (500 mg per day for 2 days) and presented with anuric AKI, low back pain, and vomiting. There was no radiographic evidence of urologic obstruction on either renal ultrasound or intravenous pyelogram, and kidney biopsy revealed tubular damage with disrupted tubular basement membranes, cellular debris, and intratubular crystalluria. By immunofluorescence, heavy deposits of Tamm-Horsfall protein (THP) were detected in Bowman’s space of more than 50% of glomeruli. The patient responded well to supportive measures and significantly recovered kidney function by day 6 of hospital admission. THP is synthesized by the thick ascending limb of Henle’s loop and is typically present in the distal segments of the nephron. Therefore, its presence in the glomerulus may have been a marker of intratubular obstruction and retrograde flow of tubular urine caused by cellular debris and crystals.The most conclusive evidence of ACZ-induced AKI, confirmed by renal biopsy, was described in 1989 by Rossert et al21 Although these patients often presented with renal colic and hematuria, AKI was not reported. For this reason, it is crucial to differentiate sulfonamide crystalluria after acute ACZ exposure as the cause of anuric AKI from calcium phosphate crystalluria most commonly attributed to chronic ACZ exposure. Therapeutic strategies should be adjusted to the specific clinical scenario. For example, sodium bicarbonate increases urine pH and would be expected to promote calcium phosphate precipitation and stone formation, but it may be useful to avoid precipitation of sulfonamide crystals .Furthermore, there are several reports of calcium phosphate kidney stones apparently induced by the chronic use of ACZ.In our patient, we hypothesize that the pathogenesis of AKI was related to ACZ-induced crystalluria causing intratubular obstruction and retrograde urine flow but not obvious intraureteric precipitation. Although ultrasonography is not sensitive enough to completely exclude intraureteric obstruction as a cause of anuric AKI, the clinical course  did not support obstructive uropathy secondary to nephrolithiasis as the primary cause of AKI.22 and adequate, timely hydration24 are required for kidney recovery, which is often observed within the first 96 hours after ACZ discontinuation. The absence of a tissue diagnosis in most of the relevant literature corresponds to the rapid recovery of kidney function in most cases. Interestingly, urine microscopy has rarely demonstrated sulfonamide crystals in most of the cases of ACZ-induced AKI, including ours. The exposure to a single dose of ketoprofen was unlikely the cause of AKI in our patient, because acute interstitial nephritis is often non-oliguric and does not typically exhibit such a rapid renal recovery. However, it is possible that ketoprofen may have decreased renal blood flow and predisposed to sulfonamide crystal toxicity. Moreover, in the absence of major risk factors for nephrolithiasis, it is feasible that repeated exposure to ACZ may have predisposed our patient to the long-term formation of kidney stones as observed on ultrasound, although no objective evidence of calcium phosphate stones was obtained.Previous case reports have clearly documented that ACZ-induced anuric AKI may present with or without radiographic obstructive uropathy. In both clinical scenarios, the resolution of the obstructionThe classic presentation of anuria and renal colic  has been described previously in patients receiving higher doses of ACZ for prolonged periods of time. However, ours is the first report of AKI after exposure to AMS prophylaxis with ACZ. Our case highlights the risk of adverse renal outcomes following ACZ ingestion even in previously healthy individuals and suggests that increased fluid intake and avoidance of nonsteroidal anti-inflammatory drugs may be advisable in travelers taking ACZ prophylaxis.


De novo replication of FMDV was first detected in oropharyngeal swab samples at 12 hpi and viremia occurred at 18–24 hpi, approximately 24 hours prior to the appearance of vesicular lesions. From 12 through 78 hpi, microscopic detection of FMDV was consistently localized to cytokeratin-positive cells within morphologically characteristic segments of oropharyngeal tonsil crypt epithelium. During this period, leukocyte populations expressing CD172a, SLA-DQ class II and/or CD8 were found in close proximity to infected epithelial cells, but with little or no co-localization with viral proteins. Similarly, M-cells expressing cytokeratin-18 did not co-localize with FMDV proteins. Intra-epithelial micro-vesicles composed of acantholytic epithelial cells expressing large amounts of structural and non-structural FMDV proteins were present within crypts of the tonsil of the soft palate during peak clinical infection. These findings inculpate the paraepiglottic tonsils as the primary site of FMDV infection in pigs exposed via the gastrointestinal tract. Furthermore, the continuing replication of FMDV in the oropharyngeal tonsils during viremia and peak clinical infection with no concurrent amplification of virus occurring in the lower respiratory tract indicates that these sites are the major source of shedding of FMDV from pigs.A time-course study was performed to elucidate the early events of foot-and-mouth disease virus (FMDV) infection in pigs subsequent to simulated natural, intra-oropharyngeal, inoculation. The earliest detectable event was primary infection in the lingual and paraepiglottic tonsils at 6 hours post inoculation (hpi) characterized by regional localization of viral RNA, viral antigen, and infectious virus. At this time FMDV antigen was localized in cytokeratin-positive epithelial cells and CD172a-expressing leukocytes of the crypt epithelium of the paraepiglottic tonsils.  Foot-and-mouth disease (FMD) is a highly contagious infection of cloven-hoofed animals, with a renowned ability of rapid transmission amongst susceptible hosts. The causative agent, FMD virus (FMDV), is a non-enveloped positive sense RNA virus belonging to the Aphthovirus genus of the Picornavirus family The characteristic clinical manifestations of FMD which include blanching and vesiculation of cornified epithelium within the oral cavity and in areas of non-haired skin, can be seen across a wide range of susceptible host-species, including domestic and wild ruminants and suids In contrast to recently gained knowledge characterizing acute FMDV-infection in cattle The aim of the current study was to elucidate the early events of FMDV pathogenesis in pigs by use of a standardized, simulated natural experimental system that has been demonstrated to efficiently and consistently infect pigs with FMDV 50) have been published previously 50/pig was used based on previous experiments confirming that this dose generates a highly synchronous infection following intra-oropharyngeal (IOP) inoculation of pigs The virus used for this study was a cattle-derived FMDV A24 Cruzeiro that had been passed once in pigs. Details regarding the generation of the virus stock, including determination of 50% pig-heel infectious doses per ml , which functions to ensure ethical and humane treatment of experimental animals. All experimental animals  were obtained from the same vendor . Following an initial clinical examination, pigs were allowed an acclimation period of approximately 1–2 weeks prior to initiation of experiments.Nineteen pigs were used for the studies described herein –2. In or50 of virus diluted to a total volume of 2 ml in minimal essential media containing 25 mM HEPES, was deposited directly onto the tonsil of the soft palate by use of a 4” stainless steel cannula. Pigs were kept with their heads in a stable position for 1 minute to ensure adequate contact between inoculum and the tonsil. As a smaller scale “proof-of-concept” study, a separate group of pigs were exposed to virus through direct contact with IOP-inoculated animals. This cohort of four pigs were housed together with two IOP-inoculated donor pigs through 24 hours. The contact exposure took place from 36 to 60 hours post inoculation (hpi) of the donors, which corresponded to the transition from pre-clinical to clinical phase of infection. The animals had free access to water during the contact period, but feed was withheld to stimulate interaction between donors and recipients. The two donor pigs were removed from the pen at the end of the 24 hour exposure.Pigs were inoculated using a novel, simulated natural system of direct intra-oropharyngeal (IOP) inoculation A standardized protocol for sampling and clinical evaluation was followed throughout the experiments. Blood samples were collected from the jugular vein, nasal swabs were obtained using small cotton swabs, and oropharyngeal (OP) swabs were obtained through direct targeting of the tonsil of the soft palate using a larger cotton swab. Swabs were immersed in 2 ml minimal essential media containing 25mM HEPES directly upon collection. Blood samples were separated through centrifugation, and OP swabs were centrifuged in order to extract the fluid absorbed by the larger cotton swab. All samples were immediately frozen at −70°C until further processing. The progression of the clinical infection (lesion distribution) was quantitated using a previously described scoring system In order to monitor acute phase infection dynamics and development of clinical disease, IOP-inoculated pigs were intensively sampled and clinically evaluated through the first 96 hpi. From these animals, blood samples were collected at 6 hour intervals from 0 to 24 hpi, and at 12 hour intervals subsequently. Clinical evaluations and collection of nasal and OP swabs were performed directly before and after inoculation and subsequently at 2 hour intervals from 4 to 12 hpi, at 3 hour intervals from 12 to 24 hpi, and at 12 hour intervals thereafter. Additionally, clinical examinations, including rectal temperature measurements, were performed at 4 hour intervals from 36 to 54 hpi in two IOP-inoculated animals in order to more precisely determine the time point at which vesicular lesions could first be observed. The 4 contact-exposed pigs were monitored through 48 hours post contact (hpc). Blood samples were collected at 12, 24 and 48 hpc, with clinical examinations and collection of swab samples performed at 4 hour intervals from 0 to 12 hpc, and at 24 and 48 hpc subsequently.Pigs were euthanized at 6, 12, 24, 48 and 78 hpi see –2 by exsEach tissue sample was divided into three 30 mg aliquots which were placed in individual tubes before being frozen on liquid nitrogen. An adjacent specimen was collected from each tissue sample, that was embedded in optimal cutting temperature media  and frozen above a bath of liquid nitrogen. Frozen tissue samples were transferred to the lab within two hours after collection, and were stored at −70°C until further processing.Two aliquots of each tissue sample collected at necropsy were individually thawed and macerated using a TissueLyser bead beater . 50 µl of tissue macerate was transferred to 96-well plates, and samples were subjected to qRT-PCR analysis following the protocol described below.10 GCN/ml for all animals sampled at each time point.Tissue macerates, serum and swabs were analyzed using qRT-PCR, targeting the 3D region of the FMDV genome Aliquots of macerated tissue samples and fluid extracted from OP swabs (see descriptions above) were cleared from debris and potential bacterial contamination by centrifugation through Spin-X filter columns (Costar Cat.no 8163). Tissue macerates, tonsil swabs and sera were subsequently analyzed for infectious FMDV through virus isolation (VI) on LFBK αvβ6 cells Following screening for contents of FMDV RNA using qRT-PCR, subsequent detection of antigen in cryosections was performed by immunohistochemistry (IHC) and multichannel immunofluorescence (MIF) as previously described Differences in positive FMDV detection between distinct tissue samples obtained from pre-viremic pigs were compared using a 2-tailed Z-test for proportions The clinical progression of FMD was highly synchronous across IOP-inoculated and contact exposed pigs that were kept alive long enough to develop clinical disease. In both IOP inoculated and contact exposed pigs, there was a consistent reduction in the activity level of the pigs observed at12 hours post exposure (hpe) when the animals became less responsive to external stimuli and more reluctant to rise. This change in clinical condition progressed through to 24 hpe, when pigs were moderately obtunded and would not rise unless strongly encouraged. Vesicular lesions were first detected in the coronary bands of the hind feet, and were observed as early as 36 hpe in one animal. At 48 hpe, all animals had coronary band vesicles on one or more feet, and rectal temperatures above 40°C. At this time point, the majority of animals were severely obtunded, with markedly reduced responses to external stimuli. Despite only moderate vesiculation at the coronary bands, several pigs were unable or unwilling to rise and/or to support their body weight while standing. The most severely affected individuals were euthanized due to animal welfare concerns. Lesion scores progressed substantially from 48 to 96 hpe , with adHigh levels of FMDV RNA, interpreted as residual inoculum, were consistently detected in OP swabs collected immediately following IOP inoculation . DetectiInfection dynamics in the 4 contact-exposed pigs were monitored following a less intensive sampling approach. These animals cohabitated with 2 IOP-inoculated donors for a period of 24 hours  of primary viral replication, the prevalence of virus detection, for both qRT-PCR and virus isolation, was determined across all pre-viremic pigs (5 pigs euthanized at 6 and 12 hpi). The highest detection prevalence was found in the paraepiglottic tonsil, with FMDV RNA detected in 4 out of 5 pigs (80%), and infectious virus in all 5 pigs investigated (100%). The detection prevalence for both viral RNA and infectious virus was 60% for the lingual tonsil, whilst the corresponding prevalence for the tonsil of the soft palate was 20%. The differences in proportions of positive detection were statistically significant between the paraepiglottic tonsil and the tonsil of the soft palate .10 RNA copies/µl and 2.69 log10 RNA copies/µl, for pigs 257 and 256, respectively; see There was marked dissemination of virus distribution coincident with detection of viremia and appearance of vesicular lesions . The two10 GCN/mg).Animals euthanized at 48 and 78 hpi were highly viremic ; Table 210 RNA copies/µl higher in the contact exposed pigs than in pigs subjected to IOP inoculation. In contact exposed pigs euthanized at 24 hpc, infectious FMDV was also isolated from a broader distribution of tissues compared to the corresponding time following IOP inoculation  and non-structural (3D) FMDV proteins were detected within crypts of the paraepiglottic tonsil . DetectiAt 48 and 78 hpi, during high titer viremia and peak clinical infection, structural and non-structural FMDV proteins were detected in segments of reticular crypt epithelium in the tonsil of the soft palate, that were morphologically similar to the regions in which virus was detected in paraepiglottic tonsils at earlier timepoints. At these times, FMDV antigens were no longer detectable in paraepiglottic tonsils. Through these later time points, there was a progressive development of intra-epithelial microvesicles expanding through the basal layers of crypt epithelium. Individual microvesicles at 48 hpi  coalesceDespite repeated IHC-screening of large quantities of tissue sections derived from other organs including lungs, skin, lymph nodes and visceral organs intracellular FMDV protein was not detected in any tissues other than the tonsil of the soft palate and peripheral vesicular lesions during peak clinical infection.The specific events defining the early pathogenesis of FMD in pigs have remained elusive despite several investigations targeting the subject. Previous works have provided valuable insight into the infection dynamics of FMDV in pigs In pre-viremic pigs, detection of FMDV by qRT-PCR and virus isolation was restricted to the pharynx. It is noteworthy that, despite inoculum being deposited directly onto the tonsil of the soft palate, the detection prevalence of virus in pre-viremic pigs was significantly higher in the paraepiglottic tonsils when compared to the tonsil of the soft palate . The paraepiglottic tonsils are small aggregates of tonsillar follicles located bilaterally within a mucosal fold at the base of the epiglottis in situ, as well as evidence of these cells supporting low levels of FMDV replication in vitroMicroscopically, the earliest detectable event in FMD pathogenesis in pigs was immunolocalization of FMDV antigen to the crypt epithelium of the paraepiglottic tonsils at 6 hpi. Individual cells containing FMDV structural and non-structural proteins were found within distinct regions of reticular epithelium within tonsillar crypts. At this early stage of infection, in contrast to later time points, FMDV antigens were also identified in a small subset of cells expressing CD172a, which is a surface marker of non-lymphoid leukocytes including both neutrophils and several classes of antigen-presenting cells of monocyte lineage Pigs euthanized at 24 hpi were in the early phase of viremia, but had not yet developed vesicular lesions. Viral distribution determined by qRT-PCR and virus isolation showed a general pattern of viral detection in pharyngeal tissues (oropharynx and nasopharynx) with additional detection in the lungs in a subset of animals. It is noteworthy that in the two IOP-inoculated pigs euthanized during this early stage of viremia, the FMDV RNA contents of the majority of pharyngeal tissues sampled, including the paraepiglottic tonsils, exceeded viral RNA levels in serum. The consistently high quantities of FMDV detected at 24 hpi in IOP-inoculated pigs further support the role of the paraepiglottic tonsil in the early pathogenesis of FMD in pigs.Immuno-microscopy of tissues harvested at 24 hpi confirmed presence of intracellular FMDV in paraepiglottic tonsils by detection of both structural (VP1) and non-structural (3D) FMDV protein in crypt epithelium. At this stage of infection, larger foci of FMDV-positive cells had spread laterally and superficially within the crypt epithelium, but without causing consistent structural disruption of the tissue. Cytokeratin-18 positive epithelial cells were consistently detected within the tonsillar crypt epithelium, but without co-localization with viral antigen. Cytokeratin-18 is a phenotypic marker of M-cells; a sub-population of specialized epithelial cells that are involved in active uptake of luminal antigen for subsequent presentation by the host immune system Pigs euthanized at 48 and 78 hpi were highly viremic and were at the peak of shedding and progression of the clinical FMD infection. At these time points serum FMDV RNA contents were similar to or higher than levels measured in the majority of sampled tissues, with exception of vesicular lesions. The highest mean quantities of FMDV RNA in tissues were found in coronary band skin, which have also previously been identified as sites supporting substantial viral amplification FMDV causes a lytic infection with substantial vesiculation of cornified epithelium at sites of secondary replication Microscopic erosions with morphologic characteristics similar to the tonsillar micro-vesicles described in the current study have previously been detected at sites of early FMDV infection in the bovine nasopharynx However, there are also clear differences between the output of the current work and what is known about the early pathogenesis of FMDV in cattle. The current localization of the primary site of FMDV infection to the oropharynx of pigs is consistent with the conventional wisdom that pigs are more susceptible to FMDV infection via the oral route in contrast to cattle, which are more likely to become infected via the respiratory route. The precise mechanisms that dictate these differences are complex and, surely, multifactorial; however, there are distinct anatomical and physiological differences between the upper respiratory and gastrointestinal tracts of cattle and pigs which may be contributing factors. Paraepiglottic tonsils, demonstrated herein as the earliest site of infection in pigs, do not exist in cattle. Interestingly, sheep have paraepiglottic tonsils and recent work in our laboratory suggests that they are involved in early pathogenesis of FMD in that species . Additionally, the nasopharyngeal mucosa of pigs tends to have less MALT and more stratified squamous epithelium compared to cattle  which may explain the apparent decreased tropism of FMDV for this region in pigs. Lastly, it may also be relevant that rumination, with repeated regurgitation of acidic rumen contents into the bovine oral cavity could decrease the viability of pH-sensitive FMDV, thus favoring infection at nasopharyngeal sites with more stabile pH.FMDV replication in the bovine nasopharynx (primary infection site) was found to decrease concurrent to establishment of viremia, whilst increasing viral replication was simultaneously detected within the lungs The overall conclusions of the work presented herein are that after infection via the upper gastrointestinal tract (oropharynx), primary FMDV replication in pigs takes place within reticular epithelium in distinct regions of oropharyngeal tonsils, with consistent predilection for the paraepiglottic tonsil. During viremia and clinical infection, substantial viral amplification occurs at secondary lesion sites with the highest viral loads found in coronary band vesicles. However, oropharyngeal tonsillar epithelium supports continued virus replication through the clinical phase of infection without detectable virus replication in the respiratory tract. Thus, it is concluded that sustained virus replication within porcine oropharyngeal tonsils, but not the lungs, contributes a major source of contagion from FMDV-infected pigs.


Alveolar macrophages (AMs) represent a unique leukocyte population that responds to airborne irritants and microbes. This distinct microenvironment coordinates the maturation of long-lived AMs, which originate from fetal blood monocytes and self-renew through mechanisms dependent on GM-CSF and CSF-1 signaling. Peripheral blood monocytes can also replenish lung macrophages; however, this appears to occur in a stimuli specific manner. In addition to mounting an appropriate immune response during infection and injury, AMs actively coordinate the resolution of inflammation through efferocytosis of apoptotic cells. Any perturbation of this process can lead to deleterious responses. In chronic obstructive pulmonary disease (COPD), there is an accumulation of airway macrophages that do not conform to the classic M1/M2 dichotomy. There is also a skewed transcriptome profile that favors expression of wound-healing M2 markers, which is reflective of a deficiency to resolve inflammation. Endogenous mediators that can promote an imbalance in inhibitory M1 vs. healing M2 macrophages are discussed, as they are the plausible mechanisms underlying why AMs fail to effectively resolve inflammation and restore normal lung homeostasis in COPD. Macrophages are essential for pulmonary host defense through their capacity to survey the exposed airways and regulate innate and adaptive immunity. The pulmonary macrophage system consists of several different populations that are found in anatomically distinct compartments, including the airways, alveolar spaces [alveolar macrophages (AMs)], and resident lung tissue. AMs constitute over 90% of the pulmonary macrophage population  and haveResident AMs are constantly encountering inhaled substances due to their exposed position in the alveolar lumen. AMs are considered to be major effector cells in innate host defense against inhaled irritants by virtue of their phagocytic ability . Therefo2+Gr1hiCX3CR1lo is actively recruited to inflamed tissues by virtue of their recognition of CCL2    . Mouse mX3CR1lo) . MonocytcR), complement, β-glucan, mannose, and several types of scavenger receptors that together facilitates phagocytosis  colonization and systemic invasion . COPD is a major global health problem and has been predicted to become the third largest cause of death in the world by 2020 . CigaretStudies have highlighted that macrophages play a pivotal role in the pathophysiology of COPD . There iChronic obstructive pulmonary disease subjects can also be very susceptible to bacterial colonization , 50 and l-arginine availability to iNOS. Reduced l-arginine bioavailability also stimulates iNOS to simultaneously produce NO and superoxide, which facilitates rapid formation of peroxynitrite (In addition to oxidative post translational modification of the host phagocytic machinery, the complex milieu within COPD airways can alter the phenotype of highly plastic airway macrophages. It is becoming increasing clear that different macrophage subpopulations exist in the inflamed lung. Although the existence of such populations is implicated in COPD, the importance of these subpopulations is unknown . The ongynitrite . Consistynitrite . Hence, + T cells, TH17, and iBALT formation are implicated in COPD pathology, the role of macrophages in regulating T cell biology remains to be elucidated.The relative balance between these polarization states can in turn, have a profound impact on disease progression [reviewed in Ref. ]. There 4 (H17 polarizing cytokines, IL-6, and IL-1β in monocyte-derived macrophages, and neutralization of IL-17A expression suppressed neutrophil airway inflammation stimulated by SAA (The accumulation of M2 skewed airway macrophages may be reflective of deficient resolution processes that normally switch off inflammation and restore lung homeostasis. Since the stimulation of non-phlogistic phagocytosis is essential to resolution of inflammation, the oxidant-dependent impairment of efferocytic clearance of damaged tissue may maintain M2 macrophages in COPD. The induction of CD163 is commonly recognized as a marker for M2-alternatively activated macrophages involved in wound-healing , 82 and 4 –90, whicd by SAA . Hence, In this review, the origin and maintenance of AMs and their essential role in innate immunity to respiratory pathogens are discussed. There is emerging evidence for the self-renewal of AMs and lung tissue macrophages through mechanisms dependent on GM-CSF and CSF-1 signaling. The role of peripheral monocytes in replenishing lung macrophages appear to be context specific, as radiation-induced lung injury stimulates monocytic replenishment, whereas influenza infection replenishes AMs through local proliferation of resident macrophages. In diseases such as COPD where there is an accumulation of airway macrophages, the relative contribution of monocyte versus local proliferation of mature AM populations remains to be determined. Lineage tracing of monocytes/macrophages in experimental models of COPD will inform on the origin of macrophages associated with disease pathology.Alveolar macrophages coordinate the efficient clearance of inhaled irritants and microbes to resolve inflammation. In addition, AMs display efferocytic activity to clear damaged tissue and cells following injury and infection. In COPD, the persistence of inflammation and the inability to efficiently clear damaged tissue and exhausted immune cells such as neutrophils may be due to excessive oxidative stress that impairs the phagocytic capacity of AMs. Airway macrophages in COPD also display a unique phenotype that is associated with the induction of M2-related genes, which are likely to be upregulated in response to local tissue damage Figure . The maiThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A strong causal association has become evident between Zika virus (ZIKV) infection during pregnancy and the occurrence of fetal growth restriction, microcephaly and eye defects. Circumstantial evidence is presented in this paper in support of the hypothesis that these effects, as well as the Guillain-Barré syndrome, are due to an endogenous form of hypervitaminosis A resulting from ZIKV infection-induced damage to the liver and the spillage of stored vitamin A compounds (“retinoids”) into the maternal and fetal circulation in toxic concentrations. Retinoids are mainly stored in the liver (about 80%) and are essential for numerous biological functions. In higher concentration, retinoids are potentially cytotoxic, pro-oxidant, mutagenic and teratogenic, especially if sudden shifts occur in their bodily distribution. Although liver involvement has not been mentioned specifically in recent reports, conventional liver enzyme tests underestimate the true extent of liver dysfunction. The proposed model could be tested by comparing retinoid concentration and expression profiles in microcephalic newborns of ZIKV-infected mothers and nonmicrocephalic newborn controls, and by correlating these profiles with measures of clinical severity. This paper presents the hypothesis that the pathogenesis of ZIKV infection-associated embryopathy involves cholestatic liver damage in early pregnancy and the spillage of stored vitamin A compounds into the circulation in toxic concentrations.Zika virus (ZIKV) 5Thirty-five cases of microcephaly were described following an outbreak of ZIKV infection in northeast Brazil in early 2015. Most of the mothers of infants with microcephaly (74%) reported a rash during the first or second trimester. During their pregnancy, all had resided in or traveled to areas where ZIKV was circulating. Most of the infants had severe microcephaly, with widespread brain calcifications in the periventricular, parenchymal and thalamic areas and basal ganglia. Ventricular enlargement with cortical/subcortical atrophy was also reported, suggesting cerebral growth arrest.6By January 2016, a total of 3530 infants with suspected microcephaly had been reported, many born to women who lived in or had visited areas of ZIKV transmission. Based on the peak number of reported cases of microcephaly in Brazil, and an assumed average duration of pregnancy of 38 weeks, the first trimester of pregnancy was temporally associated with reports of cases of febrile rash compatible with ZIKV disease in pregnant women.in utero growth restriction with or without microcephaly, five fetuses with ventricular calcification or other CNS lesions, and four with abnormal cerebral and umbilical arterial blood flow.7 Microcephaly in this cohort was part of an overall symptom profile of fetal growth restriction, cerebral calcification and eye problems that resembled rubella infection. These symptoms included pruritic rash, arthralgias, lymphadenopathy with low-grade fever in mothers, and severe growth restriction with microcephaly in the fetus. The typical clinical presentation of ZIKV infection has also been likened to mild dengue fever, but without hemorrhagic fever or death.8 The main associated clinical features of ZIKV are low-grade fever, maculopapular rash and conjunctivitis lasting 2–7 days.9A prospective study of 88 pregnant women in whom a recent rash had developed found that 82% tested positive for ZIKV, with a descending pruritic and macropapular rash, conjunctival involvement and lymphadenopathy. Fetal ultrasound detected abnormalities in 29% of the ZIKV-positive women and none in the controls. Adverse findings included two fetal deaths, five cases of n = 270,000 in 2013). Of eight cases of microcephaly reported, seven clustered around the end of the outbreak, the period of highest risk being the first trimester of pregnancy, yielding an estimated risk of microcephaly of about 1%.10 However, the rate of other abnormalities and forms of brain damage could be much higher in ZIKV-associated pregnancies, possibly comparable to that of the congenital rubella syndrome, which ranged from 38% to 100% of mothers infected in the first trimester of pregnancy.11 ZIKV therefore represents a major public health problem because of the high rate of infection in the community. Substantial evidence thus indicates that ZIKV can be transmitted from mother to fetus during pregnancy; for example, ZIKV RNA has been identified in the amniotic fluid of mothers whose fetuses had cerebral abnormalities, as shown by ultrasonography; and both viral antigen and RNA have been identified in brain tissue and placentas of children who were born with microcephaly and died soon after birth.1A causal association between ZIKV infection and microcephaly is further supported by a report on a large zika outbreak in French Polynesia. As many as 66% of the population was estimated to be infected . Bilateral abnormalities were found in 7 of the 10 infants presenting with ocular lesions, the most common of which were focal pigment mottling of the retina, chorioretinal atrophy, and optic nerve abnormalities.19 The first report was recently published providing evidence of a causal link between ZIKV infection and Guillain-Barré syndrome.20In addition to effects on the fetus, ZIKV infection has been linked to Guillain-Barré syndrome, an acute inflammatory demyelinating polyneuropathy which typically occurs after minor viral and bacterial infections. The syndrome usually begins with tingling and weakness in the feet and legs and spreads to the upper body and arms, often evolving into paralysis over a 4-week period, with accompanying disturbances of sensation and cranial nerve function. The risk of Guillain-Barré syndrome increases with age, with a predilection for males.Circumstantial evidence is presented here in support of the hypothesis that the fetal manifestations of ZIKV infection result from an endogenous form of hypervitaminosis A due to infection-induced cholestatic liver damage in early pregnancy and the spillage of stored vitamin A compounds (collectively termed retinoids) into the maternal and fetal circulation in toxic concentrations. This process is postulated to result from interactions between the ZIKV genome and endogenous retinoids, leading to retinoic acid receptor (RAR)-induced activation of hepatic stellate cells and damage to the maternal liver; furthermore, liver damage and exposure of the fetus to excess retinoid concentrations in the early weeks of pregnancy is hypothesized to cause overall fetal growth arrest, microcephaly and other congenital anomalies.22 Retinoic acid (RA), the active form of vitamin A in most cellular systems, is a fat-signaling molecule that binds to and activates the transcription of many target genes via the RARs and the retinoid X receptors.23 Retinoids in low concentration act as growth factors, whereas higher concentrations can be cytotoxic, pro-oxidant, mutagenic and teratogenic.25 About 80% of vitamin A is stored in the liver and can last for up to 2 years.26 Sudden shifts in the bodily distribution of vitamin A would therefore be expected to result in severe toxicity.Retinoids are mainly derived from the diet and are essential for multiple biological functions.27 HIV28 and human cytomegalovirus.29 Although liver dysfunction and abnormal liver function tests have not been specifically mentioned in recent reports, ZIKV was isolated from two out of three patients with jaundice in whom infection with malaria and yellow fever virus were ruled out during an investigation of yellow fever in Eastern Nigeria.30 Conventional liver enzyme tests also underestimate the true extent of liver dysfunction.31 . Symptoms usually disappear after withdrawal of vitamin A, except for occasional liver enlargement.24The hypothesis that ZIKV induces hepatic inflammation and tissue damage via increased RAR activation is consistent with the known role of excess vitamin A in causing liver damage.37 and in the effects of vitamin A supplements or excess dietary intakes of vitamin A-containing foods such as liver.38 Symptoms of acute vitamin A poisoning include fever, arthralgia and arthritis, myalgia, headache, flu-like symptoms, conjunctival congestion, lymphadenopathy, pruritus, erythematous rash, weakness, anorexia, skin peeling and altered mental status. Less common effects include hepatosplenomegaly, miscarriage, Guillain-Barré syndrome39 and thrombocytopenic purpura.37 Likewise, bone pain, fatigue40 and headache are major symptoms of hypervitaminosis A.41Strong parallels between the manifestations of ZIKV infection and those of hypervitaminosis A can be seen in the RA syndrome associated with the treatment of acute promyelocytic leukemia with RA,42 RA acts on the cell nucleus to change the pattern of gene activity by binding to specific ligand-activated RARs. Retinaldehyde dehydrogenases catalyze the synthesis of RA from retinol and determine the spatial and temporal concentration gradients of RA required for normal embryonic development.43 If consumed or administered early in pregnancy, retinoids can also cause a wide variety of congenital defects in animals and humans, depending on the stage of gestation, dose, and route of administration,44 including fetal resorption and stillbirth. Even low intakes of vitamin A in early pregnancy (7800 μg/day) are associated with congenital malformations.45Retinoids play a vital role in embryogenesis, acting as morphogens through concentration gradients in RA.46 and growth restriction in infants47 are known effects of RA and hypervitaminosis A. Rats that were administered 14 mg/kg of all-trans-RA for 13 weeks showed signs of growth arrest, anemia, elevated serum alkaline phosphatase, bone fracture and testicular degeneration.48 Microcephaly in particular is a recognized malformation of the central nervous system associated with hypervitaminosis A in early pregnancy.49 Benke50 described two infants with microcephaly, frontal bossing, hydrocephalus, microphthalmia and small, malformed and low-set undifferentiated ears whose mothers had taken the drug isotretinoin in the first trimester of pregnancy. Recalling reports of ZIKV-associated microcephaly and calcifications in the fetal brain and placenta,12 RA regulates calcification of mammalian limb cartilage51 and excess dietary intake of vitamin A promotes heart valve calcification in vivo.52Abrupt arrest of fetal growth53 Both prenatal and postnatal exposure to isotretinoin are associated with retinopathy and optic nerve abnormalities.54 RA contributes to light-induced retinopathy in mice via plasma membrane permeability and mitochondrial poisoning, caspase activation and apoptosis.55 Mutations involving a loss of retinol dehydrogenase (RDH12) have been linked to severe retinal dystrophy, involving light-induced retinal apoptosis in cone and rod photoreceptors. RDH12 shifts the retinoid balance toward increased concentrations of retinol and decreased bioactive RA, which protects against retinaldehyde-induced cell death and correlates with reduced RA concentrations in RDH12-expressing cells. RDH12 thus acts as a regulator of RA biosynthesis, protecting photoreceptors against enzymatic overproduction of RA.56With regard to eye defects, cataract is an established feature of retinoid toxicity.The hypothesis proposed here is that ZIKV infection-associated fetal growth arrest, microcephaly and related congenital anomalies, as well as the Guillain-Barré syndrome, are due to mild liver damage and resulting perturbations in retinoid metabolism during the critical period of embryogenesis. The hypothesis could be tested by comparing retinoid concentration and expression profiles in microcephalic newborns of confirmed ZIKV-infected mothers and nonmicrocephalic newborns, and by correlating these profiles with measures of clinical severity.7 Subject to testing, the hypothesis suggests the possibility that increased risks of adverse pregnancy outcomes could be detected earlier in pregnancy through serum retinoid profiling. Based on such data, it may be possible to reduce the risk of ZIKV-associated adverse clinical outcomes, including fetal growth arrest and microcephaly, by lowering circulating concentrations of retinoids. For instance, plasma retinol and its transporter RBP can be reduced by phlebotomy and/or plasmapheresis.58 While the safety of these procedures in early pregnancy is not well defined, their judicious use could potentially reduce risks of adverse fetal outcomes as well as the more severe clinical features of ZIKV infection.Microcephaly and other fetal abnormalities can be detected at 18–20 weeks of gestation by ultrasonography.


Angiogenesis, the formation of new blood vessels, is a unique and crucial biological process occurring during both development and adulthood. A better understanding of the mechanisms that regulates such process is mandatory to intervene in pathophysiological conditions. Here we highlight some recent argument on new players that are critical in endothelial cells, by summarizing novel discoveries that regulate notorious vascular pathways such as Vascular Endothelial Growth Factor (VEGF), Notch and Planar Cell Polarity (PCP), and by discussing more recent findings that put metabolism, redox signaling and hemodynamic forces as novel unforeseen facets in angiogenesis. These new aspects, that critically regulate angiogenesis and vascular homeostasis in health and diseased, represent unforeseen new ground to develop anti-angiogenic therapies. Angiogenesis is defined as the process of sprouting new blood vessels from preexisting vasculature. Neo-vessel formation is required for many physiological processes, such as embryogenesis, cardiovascular maturation, tissue repair and regeneration  in tip and stalk cells. Tip cells form filopodia that invade surrounding tissue, leading the path of neo-vessel formation  are expressed strongly in tip cells, leading to activation of VEGF pathway in these cells , leading to recruitment of mural cell  that promotes efflux of cholesterol from the membrane to High-Density-Lipoprotein (HDL) inhibits VEGF signaling and sprouting angiogenesis  that forms Low-Density-Lipoprotein (LDL) induces downregulation of VEGFR1 a decoy receptor that sequesters VEGF from VEGFR2 binding, suggesting that ApoB could activate VEGF signaling  of the key glycolysis enzyme Phosphofructokinase-2/Fructose-2,6-Bisphosphatase-3 (PFKFB3) impaired tip cell formation by interfering with Notch blockade. Overexpression of PFKB3 overcame the pro-stalk activity of Notch, while treatment with PFKB3 inhibitor, 3-(3-Pyridinyl)-1-(4-Pyridinyl)-2-Propen-1-One (3PO) mimicked the phenotype of PFKB3 KD  reduces GLUT1 expression when free lipids are available. Reduced glucose entry in the VEGF secreting cells causes a decrease of the level of the Krebs cycle intermediate alpha-Ketoglutarate . Low alpha-KG levels would promote transcription and secretion of VEGF-A  and redox events are not just passive events but can actually play a key role during angiogenesis  as well as other compounds . Rac1 promotes production of ROS via the NADPH oxidase Nox2  induced transcription of VEGF-A, by increasing transactivating activity of Specific protein 1 (Sp1) (Gonzalez-Pacheco et al., In addition, links between redox signaling and VEGF secretion were described. VEGF-A promoter presents an oxidative stress response element; and oxidative stress (via HBesides delivering oxygen and nutrients to the tissues, blood flow plays crucial roles in angiogenesis by generating frictional force that develops between flowing blood and the vascular endothelium. ECs covering the inner surface of blood vessels are constantly exposed to different types of shear stress. Shear stress is pulsatile in normal physiology, but can be oscillatory in pathologies such as atherosclerosis, profoundly affecting endothelial function and morphology (Ando and Yamamoto, The mechanism regarding how hemodynamic forces regulate vascular homeostasis is started to be decoded by the identification of the role of Piezo1 in vascular architecture Figure . Piezo pPrimary cilia are microtubule-based structures present on most mammalian cells that are important for intercellular signaling. Cilia are present on ECs where they project into the flow compartment of a blood vessel. Cilia would act as mechanical sensors of blood flow and modify the response of ECs to biomechanical forces and shear stress Figure . Two recin vitro; while its genetic deletion or pharmacological inhibition reduces vascular growth in vivo (Michalik et al., 2+, when compared to senescent/ adult endothelial cells (Moccia et al., Due to space limitation we were not able to provide a full coverage of the novel and promising mechanisms important for angiogenesis. However, we have to mention that non-coding RNA-mediated mechanisms are essential in angiogenesis. Recent work from different groups have proposed the role of exosome in regulating angiogenesis via microRNA delivery (Das and Halushka, in vivo metabolite tracers are needed to understand these mechanisms spatially and temporally. Integrative and system biology will also be of big help to understand how angiogenic signal networks are regulated in physiology and pathology, dynamically and spatially. We believe that these innovative technologies combined to the novel approaches that have displayed in the papers described in this review, will lead to important advances in the field in the coming years.All these novel findings indicate that we are shifting from a very simple model for angiogenesis, and starting to realize that metabolism, redox signaling, mechanical forces are all crucial for vascular morphogenesis. The next challenges for researchers will be to uncover the functional link among all these conditions in health and diseased conditions. This is even more exciting and intriguing considering that all these different elements are connecting with the pathways classically known to regulate angiogenesis such as VEGF or Notch signaling. Novel technologies, such as redox and metabolic sensors or RS and MS draft and write the manuscript. RS did all figures and legends. MS contribute to polish the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer PP declared a shared affiliation with the author MS and states that the process nevertheless met the standards of a fair and objective review.


Serial surveillance renal allograft biopsies have shown that early subclinical inflammation constitutes a risk factor for the development of interstitial fibrosis. More recently, it has been observed that persistent inflammation is also associated with fibrosis progression and chronic humoral rejection, two histological conditions associated with poor allograft survival. Treatment of subclinical inflammation with steroid boluses prevents progression of fibrosis and preserves renal function in patients treated with a cyclosporine-based regimen. Subclinical inflammation has been reduced after the introduction of tacrolimus based regimens, and it has been shown that immunosuppressive schedules that are effective in preventing acute rejection and subclinical inflammation may prevent the progression of fibrosis and chronic humoral rejection. On the other hand, minimization protocols are associated with progression of fibrosis, and noncompliance with the immunosuppressive regime constitutes a major risk factor for chronic humoral rejection. Thus, adequate immunosuppressive treatment, avoiding minimization strategies and reinforcing educational actions to prevent noncompliance, is at present an effective approach to combat the progression of fibrosis. Progressive renal fibrosis, regardless of the underlying aetiology, is the final common manifestation of a wide variety of chronic kidney diseases (CKD) that lead to end-stage renal disease. Fibrosis is a process of normal wound healing and repair that is activated in response to injury to maintain the original tissue architecture and functional integrity. However, prolonged chronic injurious stimuli may cause deregulation of normal processes and result in an excess deposition of extracellular matrix (ECM) . Continuβ 1 (TGF-β1) in fibrosing organs. T and B lymphocytes are also recruited to the site of injury and further facilitate secretion of fibrogenic cytokines [β1 is a potent chemoattractant for cells of macrophage-monocytic lineage. In addition to TGF-β1, monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1 (MIP-1), and macrophage inflammatory protein-2 (MIP-2) are also involved in recruitment of inflammatory cells [The pathogenesis of inflammation is complex and multifactorial, involving the interaction of cytokines, chemokines, and adhesion molecules. The participation and interaction of infiltrated cells with different cell types in the kidney is required to promote renal fibrosis. Depending on the aetiology of renal injury, tubular, glomerular, or interstitial infiltrated inflammatory cells become activated and produce fibrogenic and inflammatory cytokines. Inflammatory infiltrates, including neutrophils, macrophages, and lymphocytes, are evident in experimental models of renal disease and human renal biopsy specimens . Activatytokines . At the ry cells . The graβ superfamily are the most extensively studied growth factors that have been linked to renal fibrosis [β at different stages during the development of renal fibrotic lesions [Members of the TGF-fibrosis . Macroph lesions . However lesions , 16. Mac lesions .β and its downstream Smad signalling play an essential role. Upregulation of TGF-β is a universal finding in virtually every type of CKD, both in animal models and in humans. Despite the well documented role of TGF-β in renal fibrosis, long-term inhibition of TGF-β action, in an attempt to hamper the progression of renal fibrosis, does not seem to be an optimal approach provided that TGF-β is also an anti-inflammatory cytokine. The profibrotic and anti-inflammatory properties of TGF-β pose a dilemma for the therapeutic application of TGF-β inhibition and this is one of the reasons that novel antifibrotic targets are under active investigation [Although different fibrogenic factors have been documented, including various cytokines and hormonal, metabolic, and hemodynamic factors, it is widely accepted that TGF-tigation .β production by various cells including renal tubular cells and fibroblasts and several studies have demonstrated that the use of either AngII receptor (AT1 and AT2) antagonists or angiotensin converting enzyme (ACE) inhibitors in experimental renal disease models reduces TGF-β production and attenuates renal interstitial fibrosis [In renal fibrosis, the activation of the renin-angiotensin-aldosterone system and its main effector angiotensin II (AngII) stimulates vascular inflammation, upregulation of reactive oxygen species, cytokines, chemokines, and growth factors, and recruitment of infiltrating cells into the kidney , 20. Thefibrosis , 22.κB (nuclear factor kappa-light-chain-enhancer of activated B cells), and proinflammatory gene overexpression [κB pathway has also shown the prevention of inflammation in experimental renal damage. These observations emphasize the importance of treatments that block the AngII-induced inflammatory process in human renal diseases and provide a rationale to investigate further the involvement of the AT2/NF-κB pathway in the inflammatory response in kidney diseases [In human kidney diseases, the activated renal renin-angiotensin system has been described. In diabetic nephropathy, elevated AngII generation did correlate with the presence of inflammatory cell infiltration, the activation of NF-pression . The inhdiseases .γ-induced, cytotoxic T lymphocyte-associated, and acute rejection signatures. Pathway analysis of microarray data for the interstitial fibrosis and inflammation group also provided evidence of active participation of a range of immunologic cell types, including T cells, B cells, monocyte/macrophages, dendritic cells, and natural killer cells. Results of this study indicate that early surveillance histology with or without targeted molecular analysis provides important prognostic information. It has been suggested that analysis of intragraft innate and adaptive immune pathways during early posttransplantation years may provide the basis for early interventions aimed at altering rejection-like inflammation improving long-term survival of kidney allografts [Inflammation has also been pinpointed as a hallmark for renal transplant functional decline. Inflammation, especially when it is associated with fibrosis in surveillance kidney biopsies, is a risk factor for long-term transplant failure. Park et al.  have sholografts –26.Despite the fact that the use of new immunosuppressants has allowed reduction in the incidence of acute rejection and an improvement of short-term results in renal transplantation, long-term graft survival has been only marginally increased . Among iThe increased utilization of the so-called expanded criteria donors during the last years, that is, donors older than 60 years or donors older than 50 years with two of the following conditions, death due to stroke and history of hypertension and serum creatinine > 1.5 mg/dL, implies that a high proportion of kidneys already display interstitial fibrosis, tubular atrophy, vascular intimal thickening, and glomerulosclerosis at the time of transplantation. The severity of these lesions is associated with delayed graft function, decreased glomerular filtration rate, and decreased allograft survival. Accordingly, different scores to evaluate the severity of preexisting damage have been proposed –32. Althβ and activation of NF-κB in allografts that developed chronic changes subsequent to the occurrence of acute tubular injury [Ischemia/reperfusion injury (IRI) is a key event in organ transplantation since restoration of blood flow to ischemic tissue exacerbates tissue damage by initiating a cascade of inflammatory events including release of proinflammatory cytokines and chemokines, recruitment of leukocytes, and activation of the complement system . Differer injury . These pr injury .Preexisting chronic donor damage can progress after transplantation due to the different immunologic and nonimmunologic insults to which the kidney is exposed. To evaluate the progression of fibrosis after transplantation different groups have performed surveillance biopsies at different time points after transplantation. From the initial reports, it became clear that chronic histological damage in the tubule-interstitial, vascular, and glomerular compartments rapidly progresses during the initial months after transplantation while renal function remains stable. In different studies it has been shown that the presence of interstitial fibrosis/tubular atrophy (IF/TA) involved about 40% of transplants at 3–6 months , 40, 50%The largest study contributing to describing the natural history of the evolution of inflammation and chronic damage in stable grafts was conducted on 120 recipients receiving simultaneous kidney-pancreas transplantation in whom near 1.000 surveillance biopsies were done during 10 years of follow-up. Most severe inflammation was already observed during the first months after transplantation and tended to decrease during the first year although the inflammation persists after the first year in a proportion of patients. At the same time, interstitial fibrosis rapidly progressed during the first months after transplant. Beyond one year, glomerulosclerosis and intimal thickening slowly progressed as well as the severity of IF/TA. The presence of severe chronic lesions was associated with declining renal function and graft failure . It has Since subclinical inflammation is indistinguishable from inflammation observed in episodes of acute cellular rejection, it was tempting to propose that treatment of subclinical inflammation with steroid boluses may improve outcome after renal transplantation. The first to test this hypothesis was Rush et al.  in an elRush et al. published in 2007 the results of a multicentre trial in which patients treated with tacrolimus, mycophenolate, and prednisone were randomized, as in his previous study, to be biopsied at 1, 2, and 3 months and treated with steroid boluses in case they presented subclinical inflammation. The control group was again not biopsied and, accordingly, not treated. There were no differences between groups in the progression of fibrosis evaluated by means of a 6- and 24-month surveillance biopsy and the evolution of renal function was also not different between groups. Most remarkably, overall incidence of subclinical inflammation was less than 10% at 1, 2, and 3 months, suggesting that treatment with tacrolimus, mycophenolate mofetil, and prednisone may efficiently prevent early inflammation . After tThe prevalence of subclinical inflammation in three-month surveillance biopsies is lower in tacrolimus than in cyclosporine treated patients –61. QuanIn a prospective trial in which patients were randomized to receive 4 different immunosuppressive schedules: cyclosporine associated with mycophenolate mofetil, tacrolimus associated with mycophenolate mofetil, cyclosporine associated with sirolimus, and tacrolimus associated with sirolimus, it was observed that regimens combining a calcineurin inhibitor with sirolimus showed a lower prevalence of acute rejection during the first year, a lower prevalence of subclinical inflammation at 1-year protocol biopsy, and less severe fibrosis evaluated by means of a surveillance biopsy at 5 years, suggesting that immunosuppressive schedules that are effective in preventing acute rejection and subclinical inflammation are also effective in preventing the progression of fibrosis . At the In the last two decades, the immunosuppressive schedule has changed from cyclosporine to tacrolimus based regimens. Thus, it is interesting to compare the prevalence of chronic lesions in surveillance biopsies obtained late after transplantation in these different periods, the cyclosporine and tacrolimus era. In 2003, in the paper published by Nankivell et al. , the preFurther support for the role of immunosuppression in the prevention of early inflammation and progression of fibrosis comes from the observation that minimization of cyclosporine treatment was associated with progression of fibrosis when evaluated by means of 3- and 12-month surveillance biopsies . SimilarTaken together, these data point out that inflammation early after transplantation is a major determinant of the progression of fibrosis, appearance of HLA donor specific antibodies, and graft outcome. On the other hand, an adequate immunosuppressive treatment, avoiding minimization strategies and reinforcing educational actions to prevent noncompliance, is at present an effective approach to combat the progression of fibrosis.


HvCBF4), dehydration-responsive element-binding factors (HvDREB1), and WRKYs (HvWRKY38), in transgenic plants resulted in higher tolerance to drought and salinity, possibly by effectively altering the expression levels of stress tolerance genes due to their higher DNA binding affinity. Na+/H+ antiporters, channel proteins, and lipid transporters can also be the strong candidates for engineering plants for tolerance to salinity and low temperatures.Barley is one of the oldest cultivated crops in the world with a high adaptive capacity. The natural tolerance of barley to stress has led to increasing interest in identification of stress responsive genes through small/large-scale omics studies, comparative genomics, and overexpression of some of these genes by genetic transformation. Two major categories of proteins involved in stress tolerance are transcription factors (TFs) responsible from the re-programming of the metabolism in stress environment, and genes encoding Late Embryogenesis Abundant (LEA) proteins, antioxidant enzymes, osmolytes, and transporters. Constitutive overexpression of several barley TFs, such as C-repeat binding factors ( Drought, salinity, high or low temperatures, frost, flooding, alkaline soil, and excess or deficiency of minerals like boron and aluminum can have significant adverse effects on agricultural production  is one of the oldest cereal crops known to be cultivated since about 10,000 years in a region located between the Nile (Egypt) and Tigris Rivers (Iraq), also including Southern Turkey, Israel, Lebanon, Jordan, and Syria. It has a natural tolerance to drought, salinity, and fungal diseases, thus making it a model organism in stress biology research. Indeed, a barley plant was shown to complete its life-cycle before using all the available soil water, even in high salt concentrations and defined as the most salt-tolerant cereal  are among the initial responses to stress mainly generated by reactive oxygen species  were shown to contribute to the regulation of Na+/K+ homeostasis in barley during high salinity stress  caused better water retention capacity of transgenic plants during dehydration, and better recovery and rehydration with increased biomass production  under the transcriptional control of the stress-inducible HVA1 promoter was shown to enhance survival and biomass production upon severe salt stress and repeated cycles of severe dehydration stress in bahiagrass , an ethylene response factor (ERF)-type TF, was shown to regulate transcriptional induction of various stress-responsive genes including PDF1.2, JR3, PR1, PR5, KIN2, and GSH1, and to confer higher seed germination and root growth with high salinity in transgenic +/H+ and K+/H+ antiporters (NHXs), sucrose transporters and amino acid transporters have important roles to keep this balance. A group of transporters including NHXs, high affinity K+ transporters (HKTs), and salt overly sensitive 1 (SOS1) have been shown to maintain intracellular ion and pH homeostasis, and also contribute to the regulation of a wide variety of physiological processes associated with growth and development  showed improved biomass production under salt stress (100 mM NaCl) probably through Na+ exclusion or accumulation of excessive Na+ in the leaves  can be toxic for plants. Over expression of the barley HvAACT1 encoding a citrate transporter enhanced the Al3+ tolerance in barley and wheat  in transgenic rice increased iron uptake from alkaline soil  in maize was more effective under drought stress, and capable to enhance shoot and root growth under salt stress when compared to transgenic plants expressing either gene, and underlined the potential of their co-expression for improvement of abiotic stress tolerance  cultivars resulted in better tolerance to osmotic  stresses compared to non-transgenic control plants  resulted in higher turf quality and lower leaf wilting under water deficiency  transformed HVA1 under the constitutive actin1 promoter  transgenic lines expressing HVA1 under the control of the 35S promoter displayed enhanced drought tolerance and increased root length (Kwapata et al., Transformation of Abiotic stress tolerant crops will probably be key for food security by helping agriculture to cope with climatic change IPCC, . Barley HvWRKY38, HvDREB1, HvSNAC1, and HvCBF4 has proven to be very effective in conferring abiotic stress tolerance to other species, and provided tolerance to multiple stresses via both ABA-dependent and -independent pathways (Table Exploiting TFs in the design of stress tolerant transgenic plants has been proposed as a more effective tool than expressing single genes (Cominelli et al., HVA1 LEA protein: in field conditions they have shown improved tolerance to salinity and drought. A less investigated group of genes from barley are membrane transporters that regulate ionic homeostasis in cells, and may have a high potential for creating cultivars with better tolerance to salinity and other mineral toxicities in various crops.Single gene transformation, however, can provide good results as indicated by the performance of wheat and rice plants expressing the barley Hordeum and be used in plant breeding efforts for stress tolerance.In conclusion, despite our knowledge toward resolving barley's high survival and adaptability in stressful environment is still limited, several stress tolerance genes have been characterized well enough to move them from basic research to implementation in crops. The wild relatives of barley can be of particular interest (Shavrukov et al., FG has outlined the idea and wrote the manuscript with ZÖ and CU. DR contributed to critical reading and edition of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.