A novel GARP humanized mouse model for efficacy assessment of GARP-targeting therapies.

Although breakthroughs have been achieved with immune checkpoint inhibitors (ICI) therapy, some tumors do not respond to those therapies due to primary or acquired resistance. GARP, a type I transmembrane cell surface docking receptor mediating latent transforming growth factor-ß (TGF-ß) and abundantly expressed on regulatory T lymphocytes and platelets, is a potential target to render these tumors responsive to ICI therapy, and enhancing anti-tumor response especially combined with ICI. To facilitate these research efforts, we developed humanized mouse models expressing humanized GARP (hGARP) instead of their mouse counterparts, enabling in vivo assessment of GARP-targeting agents. We created GARP-humanized mice by replacing the mouse Garp gene with its human homolog. Then, comprehensive experiments, including expression analysis, immunophenotyping, functional assessments, and pharmacologic assays, were performed to characterize the mouse model accurately. The Tregs and platelets in the B-hGARP mice (The letter B is the first letter of the company's English name, Biocytogen.) expressed human GARP, without expression of mouse GARP. Similar T, B, NK, DCs, monocytes and macrophages frequencies were identified in the spleen and blood of B-hGARP and WT mice, indicating that the humanization of GARP did not change the distribution of immune cell in these compartments. When combined with anti-PD-1, monoclonal antibodies (mAbs) against GARP/TGF-ß1 complexes demonstrated enhanced in vivo anti-tumor activity compared to monotherapy with either agent. The novel hGARP model serves as a valuable tool for evaluating human GARP-targeting antibodies in immuno-oncology, which may enable preclinical studies to assess and validate new therapeutics targeting GARP. Furthermore, intercrosses of this model with ICI humanized models could facilitate the evaluation of combination therapies.

Transcriptional profiles of TGF-ß superfamily members in the lumbar DRGs and the effects of activins A and C on inflammatory pain in rats.

Signaling by the transforming growth factor (TGF)-ß superfamily is necessary for proper neural development and is involved in pain processing under both physiological and pathological conditions. Sensory neurons that reside in the dorsal root ganglia (DRGs) initially begin to perceive noxious signaling from their innervating peripheral target tissues and further convey pain signaling to the central nervous system. However, the transcriptional profile of the TGF-ß superfamily members in DRGs during chronic inflammatory pain remains elusive. We developed a custom microarray to screen for transcriptional changes in members of the TGF-ß superfamily in lumbar DRGs of rats with chronic inflammatory pain and found that the transcription of the TGF-ß superfamily members tends to be downregulated. Among them, signaling of the activin/inhibin and bone morphogenetic protein/growth and differentiation factor (BMP/GDF) families dramatically decreased. In addition, peripherally pre-local administration of activins A and C worsened formalin-induced acute inflammatory pain, whereas activin C, but not activin A, improved formalin-induced persistent inflammatory pain by inhibiting the activation of astrocytes. This is the first report of the TGF-ß superfamily transcriptional profiles in lumbar DRGs under chronic inflammatory pain conditions, in which transcriptional changes in cytokines or pathway components were found to contribute to, or be involved in, inflammatory pain processing. Our data will provide more targets for pain research.

Late isocaloric eating increases hunger, decreases energy expenditure, and modifies metabolic pathways in adults with overweight and obesity.

Late eating has been linked to obesity risk. It is unclear whether this is caused by changes in hunger and appetite, energy expenditure, or both, and whether molecular pathways in adipose tissues are involved. Therefore, we conducted a randomized, controlled, crossover trial (ClinicalTrials.gov NCT02298790) to determine the effects of late versus early eating while rigorously controlling for nutrient intake, physical activity, sleep, and light exposure. Late eating increased hunger (p < 0.0001) and altered appetite-regulating hormones, increasing waketime and 24-h ghrelin:leptin ratio (p < 0.0001 and p = 0.006, respectively). Furthermore, late eating decreased waketime energy expenditure (p = 0.002) and 24-h core body temperature (p = 0.019). Adipose tissue gene expression analyses showed that late eating altered pathways involved in lipid metabolism, e.g., p38 MAPK signaling, TGF-ß signaling, modulation of receptor tyrosine kinases, and autophagy, in a direction consistent with decreased lipolysis/increased adipogenesis. These findings show converging mechanisms by which late eating may result in positive energy balance and increased obesity risk.

Identification, molecular evolution, and expression analysis of the transcription factor Smad gene family in lamprey.

Transcription factor small mothers against decapentaplegic (Smad) family SMAD proteins are the essential intracellular signal mediators and transcription factors for transforming growth factor ß (TGF-ß) signal transduction pathway, which usually exert pleiotropic actions on cell physiology, including immune response, cell migration and differentiation. In this study, the Smad family was identified in the most primitive vertebrates through the investigation of the transcriptome data of lampreys. The topology of phylogenetic tree showed that the four Smads (Smad1, Smad3, Smad4 and Smad6) in lampreys were subdivided into four different groups. Meanwhile, homology analysis indicated that most Smads were conserved with typical Mad Homology (MH) 1 and MH2 domains. In addition, Lethenteron reissneri Smads (Lr-Smads) adopted general Smads folding structure and had high tertiary structural similarity with human Smads (H-Smads). Genomic synteny analysis revealed that the large-scale duplication blocks were not found in lamprey genome and neighbor genes of lamprey Smads presented dramatic differences compared with jawed vertebrates. Importantly, quantitative real-time PCR analysis demonstrated that Smads were widely expressed in lamprey, and the expression level of Lr-Smads mRNA was up-regulated with different pathogenic stimulations. Moreover, depending on the weighted gene co-expression network analysis (WGCNA), four Lr-Smads were identified as two meaningful modules (green and gray). The functional analysis of these two modules showed that they might have a correlation with ployI:C. And these genes presented strong positive correlation during the immune response from the results of Pearson's correlation analysis. In conclusion, our results would not only enrich the information of Smad family in jawless vertebrates, but also lay the foundation for immunity in further study.

TGF-ß-mediated Endothelial to Mesenchymal Transition (EndMT) and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing.

In response to specific external cues and the activation of certain transcription factors, endothelial cells can differentiate into a mesenchymal-like phenotype, a process that is termed endothelial to mesenchymal transition (EndMT). Emerging results have suggested that EndMT is causally linked to multiple human diseases, such as fibrosis and cancer. In addition, endothelial-derived mesenchymal cells may be applied in tissue regeneration procedures, as they can be further differentiated into various cell types (e.g., osteoblasts and chondrocytes). Thus, the selective manipulation of EndMT may have clinical potential. Like epithelial-mesenchymal transition (EMT), EndMT can be strongly induced by the secreted cytokine transforming growth factor-beta (TGF-ß), which stimulates the expression of so-called EndMT transcription factors (EndMT-TFs), including Snail and Slug. These EndMT-TFs then up- and downregulate the levels of mesenchymal and endothelial proteins, respectively. Here, we describe methods to investigate TGF-ß-induced EndMT in vitro, including a protocol to study the role of particular TFs in TGF-ß-induced EndMT. Using these techniques, we provide evidence that TGF-ß2 stimulates EndMT in murine pancreatic microvascular endothelial cells (MS-1 cells), and that the genetic depletion of Snail using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene editing, abrogates this phenomenon. This approach may serve as a model to interrogate potential modulators of endothelial biology, and can be used to perform genetic or pharmacological screens in order to identify novel regulators of EndMT, with potential application in human disease.

Dual ß-Catenin and γ-Catenin Loss in Hepatocytes Impacts Their Polarity through Altered Transforming Growth Factor-ß and Hepatocyte Nuclear Factor 4α Signaling.

Hepatocytes are highly polarized epithelia. Loss of hepatocyte polarity is associated with various liver diseases, including cholestasis. However, the molecular underpinnings of hepatocyte polarization remain poorly understood. Loss of ß-catenin at adherens junctions is compensated by γ-catenin and dual loss of both catenins in double knockouts (DKOs) in mice liver leads to progressive intrahepatic cholestasis. However, the clinical relevance of this observation, and further phenotypic characterization of the phenotype, is important. Herein, simultaneous loss of ß-catenin and γ-catenin was identified in a subset of liver samples from patients of progressive familial intrahepatic cholestasis and primary sclerosing cholangitis. Hepatocytes in DKO mice exhibited defects in apical-basolateral localization of polarity proteins, impaired bile canaliculi formation, and loss of microvilli. Loss of polarity in DKO livers manifested as epithelial-mesenchymal transition, increased hepatocyte proliferation, and suppression of hepatocyte differentiation, which was associated with up-regulation of transforming growth factor-ß signaling and repression of hepatocyte nuclear factor 4α expression and activity. In conclusion, concomitant loss of the two catenins in the liver may play a pathogenic role in subsets of cholangiopathies. The findings also support a previously unknown role of ß-catenin and γ-catenin in the maintenance of hepatocyte polarity. Improved understanding of the regulation of hepatocyte polarization processes by ß-catenin and γ-catenin may potentially benefit development of new therapies for cholestasis.

Phospho-Smad3 signaling is predictive biomarker for hepatocellular carcinoma risk assessment in primary biliary cholangitis patients.

INTRODUCTION: Patients with primary biliary cholangitis (PBC) are at increased risk for development of hepatocellular carcinoma (HCC), particularly in the presence of comorbidities such as excessive alcohol consumption. Although liver fibrosis is an important risk factor for HCC development, earlier predictors of future HCC development in livers with little fibrosis are needed but not well defined. The transforming growth factor (TGF)-ß/Smad signaling pathway participates importantly in hepatic carcinogenesis. Phosphorylated forms (phospho-isoforms) in Smad-related pathways can transmit opposing signals: cytostatic C-terminally-phosphorylated Smad3 (pSmad3C) and carcinogenic linker-phosphorylated Smad3 (pSmad3L) signals. METHODS AND RESULTS: To assess the balance between Smad signals as a biomarker of risk, we immunohistochemically compared Smad domain-specific Smad3 phosphorylation patterns among 52 PBC patients with various stages of fibrosis and 25 non-PBC patients with chronic hepatitis C virus infection. HCC developed in 7 of 11 PBC patients showing high pSmad3L immunoreactivity, but in only 2 of 41 PBC patients with low pSmad3L. In contrast, 9 of 20 PBC patients with minimal Smad3C phosphorylation developed HCC, while HCC did not occur during follow-up in 32 patients who retained hepatic tumor-suppressive pSmad3C. Further, PBC patients whose liver specimens showed high pSmad3L positivity were relatively likely to develop HCC even when little fibrosis was evident. CONCLUSION: In this study, Smad phospho-isoform status showed promise as a biomarker predicting likelihood of HCC occurrence in PBC. Eventually, therapies to shift favorably Smad phospho-isoforms might decrease likelihood of PBC-related HCC.

Mechanism of threshold size assessment: Metamorphosis is triggered by the TGF-beta/Activin ligand Myoglianin.

Although the mechanisms that control growth are now well understood, the mechanism by which animals assess their body size remains one of the great puzzles in biology. The final larval instar of holometabolous insects, after which growth stops and metamorphosis begins, is specified by a threshold size. We investigated the mechanism of threshold size assessment in the tobacco hornworm, Manduca sexta. The threshold size was found to change depending on the amount of exposure to poor nutrient conditions whereas hypoxia treatment consistently led to a lower threshold size. Under these various conditions, the mass of the muscles plus integuments was correlated with the threshold size. Furthermore, the expression of myoglianin (myo) increased at the threshold size in both M. sexta and Tribolium castaneum. Knockdown of myo in T. castaneum led to larvae that underwent supernumerary larval molts and stayed in the larval stage permanently even after passing the threshold size. We propose that increasing levels of Myo produced by the growing tissues allow larvae to assess their body size and trigger metamorphosis at the threshold size.

Assessment of Hepatoprotective Effect of Chokeberry Juice in Rats Treated Chronically with Carbon Tetrachloride.

The aim of this study was to compare the protective effects of chokeberry juice and silymarin against chemical-induced liver fibrosis in rats. Liver fibrosis was induced by CCl4 administered two days a week for six weeks. Two groups of rats were co-treated with chokeberry juice, 10 mL/kg/day. or silymarin as a positive control, 100 mg/kg/day for six weeks. Hepatic lipid peroxidation was suppressed by 50% and the activity of hepatic antioxidant enzymes was increased by 19%-173% in rats co-treated with CCl4 and substances tested as compared to rats administered CCl4 alone. Hepatic hydroxyproline was decreased by 24% only in rats treated with silymarin. The messenger RNA (mRNA) expression levels of fibrosis-related molecules, procollagen I, α-SMA, TIMP-1, TGFß, and TNFα, which were significantly increased in the liver of CCl4-treated rats, were not modulated by substances tested. Histological evaluation revealed a slight protective effect of silymarin against fibrosis. However, in CCl4 + chokeberry-treated rats, the density of vacuolated hepatocytes was significantly lower than that in silymarin administered animals. Chokeberry juice did not demonstrate an antifibrotic effect in the applied experimental model of fibrosis, and the effect of the known antifibrotic agent, silymarin, was very limited.

Assessment of Bones Deficient in Fibrillin-1 Microfibrils Reveals Pronounced Sex Differences.

Defects in the extracellular matrix protein fibrillin-1 that perturb transforming growth factor beta (TGFß) bioavailability lead to Marfan syndrome (MFS). MFS is an autosomal-dominant disorder, which is associated with connective tissue and skeletal defects, among others. To date, it is unclear how biological sex impacts the structural and functional properties of bone in MFS. The aim of this study was to investigate the effects of sex on bone microarchitecture and mechanical properties in mice with deficient fibrillin-1, a model of human MFS. Bones of 11-week-old male and female Fbn1mgR/mgR mice were investigated. Three-dimensional micro-computed tomography of femora and vertebrae revealed a lower ratio of trabecular bone volume to tissue volume, reduced trabecular number and thickness, and greater trabecular separation in females vs. males. Three-point bending of femora revealed significantly lower post-yield displacement and work-to-fracture in females vs. males. Mechanistically, we found higher Smad2 and ERK1/2 phosphorylation in females vs. males, demonstrating a greater activation of TGFß signaling in females. In summary, the present findings show pronounced sex differences in the matrix and function of bones deficient in fibrillin-1 microfibrils. Consequently, sex-specific analysis of bone characteristics in patients with MFS may prove useful in improving the clinical management and life quality of these patients, through the development of sex-specific therapeutic approaches.

Ionizing Radiation Promotes Epithelial-to-Mesenchymal Transition in Lung Epithelial Cells by TGF-ß-producing M2 Macrophages.

BACKGROUND/AIM: Ionizing radiation induces pulmonary fibrosis, which is a common dose-limiting complication in patients receiving radiotherapy. Fibrosis occurs through the accumulation of large amounts of ECM components, synthesized by myofibroblasts in damaged lung tissue. Epithelial cells serve as one of the cellular sources of myofibroblasts via the epithelial-to-mesenchymal transition (EMT) process. In this study, we investigated the role of TGF-ß-secreting M2 macrophages in association with ionizing radiation-induced EMT. MATERIALS AND METHODS: The lung epithelial cell line MLE12, was irradiated and the expression of EMT markers and chemokines was examined. Moreover, the mouse lung macrophage MH-S cell line was cultured with conditioned media from irradiated MLE12 cells, to examine the effects of the secreted factors on the migration ability of macrophages. For the murine pulmonary fibrosis model, mice were locally irradiated and the levels of M1 or M2 macrophage-related markers and cytokines were measured in bronchoalvelolar lavage (BAL) fluid and lung tissue. RESULTS: In MLE12 cells, irradiation directly induced expression of EMT-related markers and secretion of various chemokines, which lead to macrophage migration. Interestingly, the sub-population of macrophages recruited in the lung of mice after thoracic irradiation was M2 macrophages that expressed Arg-1 and CD206. M2 macrophages induced the MLE12 to undergo phenotypic conversion to form fibroblast-like cells, which leads to a down-regulation of epithelial markers and an up-regulation of new EMT-related markers. In thoracic irradiated mice, pro-inflammatory cytokines such as IL-1ß, IL-4 and IL-10 were increased at 2 weeks, but returned to normal levels from 16 weeks or 24 weeks after irradiation. However, thoracic irradiation led to a rapid increase of TGF-ß and IGF-1 levels, which lasted up to 24 weeks. It was confirmed that M2 macrophages secreted the high levels of TGF-ß. Moreover, the elimination of TGF-ß from M2 macrophages attenuated mesenchymal transition of MLE12. CONCLUSION: TGF-ß-secreting M2 macrophages play an important regulatory role in mesenchymal transition of epithelial cells in the lung of irradiated mice, thus contributing to radiation-induced pulmonary fibrosis.

Assessment of the Immunosuppressive Potential of INF-γ Licensed Adipose Mesenchymal Stem Cells, Their Secretome and Extracellular Vesicles.

There is an active search for the ideal strategy to potentialize the effects of Mesenchymal Stem-Cells (MSCs) over the immune system. Also, part of the scientific community is seeking to elucidate the therapeutic potential of MSCs secretome and its extracellular vesicles (EVs), in order to avoid the complexity of a cellular therapy. Here, we investigate the effects of human adipose MSCs (AMSCs) licensing with INF-γ and TLR3 agonist over AMSCs proliferation, migration, as well as the immunomodulatory function. Furthermore, we evaluated how the licensing of AMSCs affected the immunomodulatory function of AMSC derived-secretome, including their EVs. INF-γ licensed-AMSCs presented an elevated expression of indoleamine 2,3-dioxygenase (IDO), accompanied by increased ICAM-1, as well as a higher immunosuppressive potential, compared to unlicensed AMSCs. Interestingly, the conditioned medium obtained from INF-γ licensed-AMSCs also revealed a slightly superior immunosuppressive potential, compared to other licensing strategies. Therefore, unlicensed and INF-γ licensed-AMSCs groups were used to isolate EVs. Interestingly, EVs isolated from both groups displayed similar capacity to inhibit T-cell proliferation. EVs isolated from both groups shared similar TGF-ß and Galectin-1 mRNA content but only EVs derived from INF-γ licensed-AMSCs expressed IDO mRNA. In summary, we demonstrated that INF-γ licensing of AMSCs provides an immunosuppressive advantage both from a cell-cell contact-dependent perspective, as well as in a cell-free context. Interestingly, EVs derived from unlicensed and INF-γ licensed-AMSCs have similar ability to control activated T-cell proliferation. These results contribute towards the development of new strategies to control the immune response based on AMSCs or their derived products.

Assessment of Topical Therapies for Improving the Optical Clarity Following Stromal Wounding in a Novel Ex Vivo Canine Cornea Model.

Purpose: To evaluate the effect of topical suberanilohydroxamic acid (SAHA) and 5-methyl-1-phenyl-2[1H]-pyridone (pirfenidone) on the degree of corneal haze in the stromal wounded ex vivo canine cornea. Methods: Twenty-four corneoscleral rims from normal dogs were uniformly wounded with an excimer laser and placed into culture medium with an air-liquid interface. The control group (n = 8) contained placebo-treated corneas. Treatment group 1 (n = 8) received SAHA topically every 6 hours. Treatment group 2 (n = 8) received pirfenidone topically every 6 hours. Each cornea was fluorescein stained and macrophotographed every 6 hours to assess epithelialization rate. All corneas were also macrophotographed weekly to assess optical clarity (haze). Images were analyzed for differences in pixel intensity between wounded (haze) and unwounded (nonhaze) regions, and haze surface area for each cornea was calculated. Results: The mean epithelialization time was 47.25 hours in the control group, 45.00 hours in the SAHA group, and 43.50 hours in the pirfenidone group, revealing no significant difference (P = 0.368). The median difference in pixel intensity between haze and nonhaze areas was 21.5 in the control group, 8.0 in the SAHA group, and 8.0 in the pirfenidone group, which is significant (P < 0.01). The median haze surface area was 12.96 mm2 in the control group, 5.70 mm2 in the SAHA group, and 5.92 mm2 in the pirfenidone group, which is significant (P < 0.01). Conclusions: Stromal-wounded ex vivo canine corneas exhibited greater optical clarity when treated with SAHA and pirfenidone than when placebo treated at 21 days. There was no significant difference in epithelialization rate between groups. Corneal contour was correlated with geographic haze distribution.

Methods for the Assessment of Active Transforming Growth Factor-ß in Cells and Tissues.

The potent and pluripotent cytokine TGFß has important roles in normal homeostasis and disease pathogenesis. Once released from cells, TGFß exists in both latent and functionally active forms. Large amounts of latent TGFß are secreted from cells and sequestered in extracellular matrix, only a small proportion of which is activated at any given time. Accurate assessment of TGFß activity levels is an important measurement in biological research and requires methods distinct from measuring total levels of TGFß expression as small changes in TGFß activity levels could be masked by the large amounts of latent TGFß available to be measured. In this chapter, we describe detailed experimental methods for assessing levels of active TGFß in cells and tissues. This chapter includes methods for the assessment of TGFß activity in cells in vitro, in ex vivo precision cut tissue, and in vivo.

Flow Cytometric Assessment of STAT Molecules in Th9 Cells.

IL-9-producing Th9 cells are a novel subset of T helper cells that develop independently of other T helper subsets. Th9 cells have been implicated in the pathogenesis of allergic asthma and autoimmunity, while also serving as critical effector T cells in mediating antitumor immune responses. Concomitant presence of TGF-ß and IL-4 lead to the differentiation of naïve CD4+ T cells towards the Th9 phenotype. In addition, several cytokines, including IL-1ß, IL-2, IL-25, and IL-33, further amplify Th9 responses. Negative regulators of Th9 cells include other cytokines such as IFN-γ, IL-23, and IL-27. Here, we describe a detailed protocol for the analysis of STAT molecules involved in the differentiation of Th9 cells and Th9 inhibition by IL-27.

Computational Model of Secondary Palate Fusion and Disruption.

Morphogenetic events are driven by cell-generated physical forces and complex cellular dynamics. To improve our capacity to predict developmental effects from chemical-induced cellular alterations, we built a multicellular agent-based model in CompuCell3D that recapitulates the cellular networks and collective cell behavior underlying growth and fusion of the mammalian secondary palate. The model incorporated multiple signaling pathways (TGFß, BMP, FGF, EGF, and SHH) in a biological framework to recapitulate morphogenetic events from palatal outgrowth through midline fusion. It effectively simulated higher-level phenotypes (e.g., midline contact, medial edge seam (MES) breakdown, mesenchymal confluence, and fusion defects) in response to genetic or environmental perturbations. Perturbation analysis of various control features revealed model functionality with respect to cell signaling systems and feedback loops for growth and fusion, diverse individual cell behaviors and collective cellular behavior leading to physical contact and midline fusion, and quantitative analysis of the TGF/EGF switch that controls MES breakdown-a key event in morphogenetic fusion. The virtual palate model was then executed with theoretical chemical perturbation scenarios to simulate switch behavior leading to a disruption of fusion following chronic (e.g., dioxin) and acute (e.g., retinoic acid) chemical exposures. This computer model adds to similar systems models toward an integrative "virtual embryo" for simulation and quantitative prediction of adverse developmental outcomes following genetic perturbation and/or environmental disruption.

Assessment of the Effects of Rituximab Monotherapy on Different Subsets of Circulating T-Regulatory Cells and Clinical Disease Severity in Severe Pemphigus Vulgaris.

BACKGROUND: Robust evidence for the efficacy of rituximab monotherapy in pemphigus is lacking. The effects of rituximab on T-regulatory cells (Tregs) in pemphigus have not been studied. OBJECTIVE: The primary objective was to assess the efficacy of rituximab monotherapy in severe pemphigus vulgaris. The secondary objectives were to assess whether counts of different subsets of Tregs in the peripheral blood correlate with baseline clinical severity and whether clinical response in severe pemphigus is associated with an alteration in the Treg count. METHODS: Eighteen eligible subjects with severe pemphigus vulgaris were recruited and were treated with 1 g of intravenous rituximab on days 0 and 15. Efficacy was assessed in terms of disease control, time to disease control, complete remission off therapy, and relapse. Flow cytometric analysis of CD4+CD25+FoxP3, IL-10-secreting Tr1, and TGF-ß secreting Th3 regulatory cells was performed. Clinical evaluation and flow cytometric analysis of Tregs was performed periodically until follow-up at 26 weeks. RESULTS: Rituximab monotherapy was able to induce complete remission in all but 5 (68.75%) patients and was well tolerated. No direct relationship between clinical severity and CD4+CD25+FoxP3 cell counts was found. There were inverse correlations between serially measured values of the cutaneous and mucosal Autoimmune Bullous Skin Disorder Intensity Score (ABSIS) and Th3 cell count. CONCLUSION: Rituximab is a safe and effective monotherapy option for severe pemphigus. As the immunological findings were somewhat different from those observed in other autoimmune conditions treated with rituximab, further studies are required to substantiate the findings of our study in pemphigus patients.

Histological changes and immunohistochemical markers in the assessment of glomerulosclerosis in patients with glomerulonephritis.

INTRODUCTION: Glomerular cells (mesangial, endothelial, epithelial) are activated during glomerulonephritis, a process indicated by the expression of the immunohistochemical marker α-smooth muscle actin (SMA). Many growth factors participate in the above-mentioned processes, among them of great importance is the transforming growth factor ß (TGF-ß). The result of these changes is represented by active lesions (mesangial matrix increase, mesangial cell proliferation) and chronic fibrotic lesions (glomerulosclerosis). METHODOLOGY: We studied a group of 41 patients with primary and secondary glomerulonephritis (24 males, 17 females, mean age 45.5 ± 12.9 years), which underwent kidney biopsies, processed in light microscopy. We performed immunohistochemistry procedures with monoclonal antibodies (performed with the LSAB2-HRP system: anti-α-SMA, and anti-TGF-ß), which were assessed using a semiquantitative score, that was correlated with the histological and biological data. In order to quantify the histological changes and to assess the extent of active-inflammatory and chronic-sclerotic/fibrotic lesions, we adapted a scoring system initially used only for lupus nephritis, and ANCA-associated vasculitis. RESULTS: TGF-ß expression in glomerular endothelial cells correlated with mesangial matrix increase (r=0.28, p<0.05), total activity index (r=0.29, p<0.05) and total chronicity index (r=0.34, p<0.05). Glomerular epithelial cell TGF-ß correlates with mesangial proliferation (r=0.29, p<0.05), mesangial matrix increase (r=0.4, p<0.01) and total activity index (r=0.28, p<0.05). We observed a strong correlation between endothelial immunolabeling of SMA and the mesangial proliferation score (r=-0.96, p<0.005) and also an indirect correlation with the glomerulosclerosis score (r=-0.35, p<0.05) and the total chronicity index (r=-0.39, p<0.05). Concerning biological data there was a correlation between mesangial SMA expression and serum creatinine (r=0.60, p<0.001) and an indirect correlation with GFR (r=-0.37, p<0.05). CONCLUSIONS: We conclude that TGF-ß has a key role in determining glomerulosclerosis especially through mesangial matrix increase, but possibly also through mesangial cells proliferation. Another role of this growth factor is related to transdifferentiation, not only epithelial-mesenchymal, but also endothelial-mesenchymal.