Basement membranes in the cornea and other organs that commonly develop fibrosis.

Basement membranes are thin connective tissue structures composed of organ-specific assemblages of collagens, laminins, proteoglycan-like perlecan, nidogens, and other components. Traditionally, basement membranes are thought of as structures which primarily function to anchor epithelial, endothelial, or parenchymal cells to underlying connective tissues. While this role is important, other functions such as the modulation of growth factors and cytokines that regulate cell proliferation, migration, differentiation, and fibrosis are equally important. An example of this is the critical role of both the epithelial basement membrane and Descemet's basement membrane in the cornea in modulating myofibroblast development and fibrosis, as well as myofibroblast apoptosis and the resolution of fibrosis. This article compares the ultrastructure and functions of key basement membranes in several organs to illustrate the variability and importance of these structures in organs that commonly develop fibrosis.

Fibrocyte migration, differentiation and apoptosis during the corneal wound healing response to injury.

The aim of this study was to determine whether bone marrow-derived fibrocytes migrate into the cornea after stromal scar-producing injury and differentiate into alpha-smooth muscle actin (αSMA) + myofibroblasts. Chimeric mice expressing green fluorescent protein (GFP) bone marrow cells had fibrosis (haze)-generating irregular phototherapeutic keratectomy (PTK). Multiplex immunohistochemistry (IHC) for GFP and fibrocyte markers (CD34, CD45, and vimentin) was used to detect fibrocyte infiltration into the corneal stroma and the development of GFP+ αSMA+ myofibroblasts. IHC for activated caspase-3, GFP and CD45 was used to detect fibrocyte and other hematopoietic cells undergoing apoptosis. Moderate haze developed in PTK-treated mouse corneas at 14 days after surgery and worsened, and persisted, at 21 days after surgery. GFP+ CD34+ CD45+ fibrocytes, likely in addition to other CD34+ and/or CD45+ hematopoietic and stem/progenitor cells, infiltrated the cornea and were present in the stroma in high numbers by one day after PTK. The fibrocytes and other bone marrow-derived cells progressively decreased at four days and seven days after surgery. At four days after PTK, 5% of the GFP+ cells expressed activated caspase-3. At 14 days after PTK, more than 50% of GFP+ CD45+ cells were also αSMA+ myofibroblasts. At 21 days after PTK, few GFP+ αSMA+ cells persisted in the stroma and more than 95% of those remaining expressed activated caspase-3, indicating they were undergoing apoptosis. GFP+ CD45+ SMA+ cells that developed from 4 to 21 days after irregular PTK were likely developed from fibrocytes. After irregular PTK in the strain of C57BL/6-C57/BL/6-Tg(UBC-GFP)30Scha/J chimeric mice, however, more than 95% of fibrocytes and other hematopoietic cells underwent apoptosis prior to the development of mature αSMA+ myofibroblasts. Most GFP+ CD45+ αSMA+ myofibroblasts that did develop subsequently underwent apoptosis-likely due to epithelial basement membrane regeneration and deprivation of epithelium-derived TGFß requisite for myofibroblast survival.

Epithelial basement membrane injury and regeneration modulates corneal fibrosis after pseudomonas corneal ulcers in rabbits.

The purpose of this study was to investigate whether myofibroblast-related fibrosis (scarring) after microbial keratitis was modulated by the epithelial basement membrane (EBM) injury and regeneration. Rabbits were infected with Pseudomonas aeruginosa after epithelial scrape injury and the resultant severe keratitis was treated with topical tobramycin. Corneas were analyzed from one to four months after keratitis with slit lamp photos, immunohistochemistry for alpha-smooth muscle actin (α-SMA) and monocyte lineage marker CD11b, and transmission electron microscopy. At one month after keratitis, corneas had no detectible EBM lamina lucida or lamina densa, and the central stroma was packed with myofibroblasts that in some eyes extended to the posterior corneal surface with damage to Descemet's membrane and the endothelium. At one month, a nest of stromal cells in the midst of the SMA + myofibroblasts in the stroma that were CD11b+ may be fibrocyte precursors to myofibroblasts. At two to four months after keratitis, the EBM fully-regenerated and myofibroblasts disappeared from the anterior 60-90% of the stroma of all corneas, except for one four-month post-keratitis cornea where anterior myofibroblasts were still present in one localized pocket in the cornea. The organization of the stromal extracellular matrix also became less disorganized from two to four months after keratitis but remained abnormal compared to controls at the last time point. Myofibroblasts persisted in the posterior 10%-20% of posterior stroma even at four months after keratitis in the central cornea where Descemet's membrane and the endothelium were damaged. This study suggests that the EBM has a critical role in modulating myofibroblast development and fibrosis after keratitis-similar to the role of EBM in fibrosis after photorefractive keratectomy. Damage to EBM likely allows epithelium-derived transforming growth factor beta (TGFß) to penetrate the stroma and drive development and persistence of myofibroblasts. Eventual repair of EBM leads to myofibroblast apoptosis when the cells are deprived of requisite TGFß to maintain viability. The endothelium and Descemet's membrane may serve a similar function modulating TGFß penetration into the posterior stroma-with the source of TGFß likely being the aqueous humor.

Akt-dependent enhanced migratory capacity of Th17 cells from children with lupus nephritis.

Th17 cells infiltrate the kidneys of patients with lupus nephritis (LN) and are critical for the pathogenesis of this disease. In this study, we show that enhanced activity of Stat3 in CD4(+)CD45RA(-)Foxp3(-) and Foxp3(low) effector T cells from children with LN correlates with increased frequencies of IL-17-producing cells within these T cell populations. The levels of retinoic acid-related orphan receptor c and IL-17 mRNA are significantly higher in PBMCs from children with LN than in those from controls. Mammalian target of rapamycin inhibition by rapamycin reduces both Stat3 activation in effector T cells and the frequency of IL-17-producing T cells in lupus patients. Complement factor C5a slightly increases the expression of IL-17 and induces activation of Akt in anti-CD3-activated lupus effector T cells. Th17 cells from children with LN exhibit high Akt activity and enhanced migratory capacity. Inhibition of the Akt signaling pathway significantly decreases Th17 cell migration. These findings indicate that the Akt signaling pathway plays a significant role in the migratory activity of Th17 cells from children with LN and suggest that therapeutic modulation of the Akt activity may inhibit Th17 cell trafficking to sites of inflammation and thus suppress chronic inflammatory processes in children with LN.

Genomics of Resistance to Targeted Therapies.

Targeting BCR and BCL-2 signaling is a widely used therapeutic strategy for chronic lymphocytic leukemia. C481S mutation decreases the covalent binding affinity of ibrutinib to BTK, resulting in reversible rather than irreversible inhibition. In addition to BTK, mutations in PLCG2 have been demonstrated to mediate acquired ibrutinib resistance. Venetoclax, a highly selective BCL2 inhibitor, has high affinity to the BH3-binding grove of BCL2. Mutation in BCL2 (Gly101Val) decreases the affinity of BCL2 for venetoclax and confers acquired resistance in cell lines and primary patient cells. This review discusses the common mechanisms of resistance to targeted therapies in chronic lymphocytic leukemia.

Early Intervention with Lenalidomide in Patients with High-risk Chronic Lymphocytic Leukemia.

PURPOSE: Infectious complications constitute a leading cause of morbidity and mortality in chronic lymphocytic leukemia (CLL). Patients respond poorly to vaccines, particularly pneumococcal polysaccharide and influenza vaccines. In addition, patients with genetically high-risk disease are at increased risk for early disease progression and death. Lenalidomide, an oral immunomodulatory agent with demonstrated clinical activity in CLL, can potentially restore immune system dysfunction associated with CLL while improving disease outcomes. PATIENTS AND METHODS: Phase II study randomized 49 patients with genetically high-risk CLL or small lymphocytic lymphoma [SLL; defined as unmutated Ig heavy chain variable region, deletion(17p) or (11q), and/or complex abnormal karyotype], to receive lenalidomide either concurrent (arm A) or sequential to (arm B) two doses of 13-valent protein-conjugated pneumococcal vaccine (PCV13) administered 2 months apart, in patients not meeting International Workshop on Chronic Lymphocytic Leukemia treatment criteria. RESULTS: Four serotypes (3, 4, 5, 6B) achieved the additional seroprotection definition of a fourfold increase in arm A, and six serotypes (3, 4, 5, 6B, 19A, 19F) in arm B. All patients achieved the defined concentration of 0.35 µg/mL for at least one serotype tested. No significant difference was observed with the addition of lenalidomide. At median time on treatment of 3.6 years, median progression-free survival (PFS) was 5.8 years [95% confidence interval (CI), 3.1-not reached]. PFS at 1, 2, and 3 years was 85% (95% CI, 72-93), 79% (95% CI, 64-88), and 72% (95% CI, 57-83), respectively. CONCLUSIONS: Lenalidomide is efficacious with manageable toxicities as an early intervention strategy in patients with high-risk CLL, but did not enhance humoral response to PCV13 vaccine.

Gly101Val BCL2 Mutation: One Step Closer to Understanding Venetoclax Resistance in CLL.

In this issue, Blombery and colleagues show that the chronic lymphocytic leukemia (CLL) cells bearing Gly101Val mutation confer resistance to venetoclax by reducing the affinity of BCL2 for venetoclax by 180-fold in cell lines and in patient cells. Detection of this mutation provides a potential biomarker for impending disease progression and an opportunity for targeted and combinational therapy to treat CLL.See related article by Blombery et al., p. 342.

IL-1 and TGF-ß Modulation of Epithelial Basement Membrane Components Perlecan and Nidogen Production by Corneal Stromal Cells.

Purpose: To determine whether (1) the in vitro expression of epithelial basement membrane components nidogen-1, nidogen-2, and perlecan by keratocytes, corneal fibroblasts, and myofibroblasts is modulated by cytokines/growth factors, and (2) perlecan protein is produced by stromal cells after photorefractive keratectomy. Methods: Marker-verified rabbit keratocytes, corneal fibroblasts, myofibroblasts were stimulated with TGF-ß1, IL-1α, IL-1ß, TGF-ß3, platelet-derived growth factor (PDGF)-AA, or PDGF-AB. Real-time quantitative RT-PCR was used to detect expression of nidogen-1, nidogen-2, and perlecan mRNAs. Western blotting evaluated changes in protein expression. Immunohistochemistry was performed on rabbit corneas for perlecan, alpha-smooth muscle actin, keratocan, vimentin, and CD45 at time points from 1 day to 1 month after photorefractive keratectomy (PRK). Results: IL-1α or -1ß significantly upregulated perlecan mRNA expression in keratocytes. TGF-ß1 or -ß3 markedly downregulated nidogen-1 or -2 mRNA expression in keratocytes. None of these cytokines had significant effects on nidogen-1, -2, or perlecan mRNA expression in corneal fibroblasts or myofibroblasts. IL-1α significantly upregulated, while TGF-ß1 significantly downregulated, perlecan protein expression in keratocytes. Perlecan protein expression was upregulated in anterior stromal cells at 1 and 2 days after -4.5 or -9 diopters (D) PRK, but the subepithelial localization of perlecan became disrupted at 7 days and later time points in -9-D PRK corneas when myofibroblasts populated the anterior stroma. Conclusions: IL-1 and TGF-ß1 have opposing effects on perlecan and nidogen expression by keratocytes in vitro. Proximate participation of keratocytes is likely needed to regenerate normal epithelial basement membrane after corneal injury.

The Impact of Photorefractive Keratectomy and Mitomycin C on Corneal Nerves and Their Regeneration.

PURPOSE: To determine how photorefractive keratectomy (PRK) and mitomycin C (MMC) affect corneal nerves and their regeneration over time after surgery. METHODS: Twenty-eight New Zealand rabbits had corneal epithelial scraping with (n = 3) and without (n = 3) MMC 0.02% or -9.00 diopter PRK with (n = 6) and without (n = 16) MMC 0.02%. Corneas were removed after death and corneal nerve morphology was evaluated using acetylcholinesterase immunohistochemistry and beta-III tubulin staining after 1 day for all groups, after 1 month for PRK with and without MMC, and 2, 3, and 6 months after PRK without MMC. Image-Pro software (Media Cybernetics, Rockville, MD) was used to quantitate the area of nerve loss after the procedures and, consequently, regeneration of the nerves over time. Opposite eyes were used as controls. RESULTS: Epithelial scraping with MMC treatment did not show a statistically significant difference in nerve loss compared to epithelial scraping without MMC (P = .40). PRK with MMC was significantly different from PRK without MMC at 1 day after surgery (P = .0009) but not different at 1 month after surgery (P = .90). In the PRK without MMC group, nerves regenerated at 2 months (P < .0001) but did not return to the normal preoperative level of innervation until 3 months after surgery (P = .05). However, the morphology of the regenerating nerves was abnormal-with more tortuosity and aberrant innervation compared to the preoperative controls-even at 6 months after surgery. CONCLUSIONS: PRK negatively impacts the corneal nerves, but they are partially regenerated by 3 months after surgery in rabbits. Nerve loss after PRK extended peripherally to the excimer laser ablated zone, indicating that there was retrograde degeneration of nerves after PRK. MMC had a small additive toxic effect on the corneal nerves when combined with PRK that was only significant prior to 1 month after surgery. [J Refract Surg. 2018;34(12):790-798.].

T cells in multiple myeloma display features of exhaustion and senescence at the tumor site.

BACKGROUND: Multiple myeloma is an incurable plasma cell malignancy that is mostly restricted to the bone marrow. Cancer-induced dysfunction of cytotoxic T cells at the tumor site may be responsible for immune evasion and therapeutical failure of immunotherapies. Therefore, enhanced knowledge about the actual status of T cells in myeloma bone marrow is urgently needed. Here, we assessed the expression of inhibitory molecules PD-1, CTLA-4, 2B4, CD160, senescence marker CD57, and CD28 on T cells of naive and treated myeloma patients in the bone marrow and peripheral blood and collected data on T cell subset distribution in both compartments. In addition, T cell function concerning proliferation and expression of T-bet, IL-2, IFNγ, and CD107a was investigated after in vitro stimulation by CD3/CD28. Finally, data was compared to healthy, age-matched donor T cells from both compartments. METHODS: Multicolor flow cytometry was utilized for the analyses of surface molecules, intracellular staining of cytokines was also performed by flow cytometry, and proliferation was assessed by 3H-thymidine incorporation. Statistical analyses were performed utilizing unpaired T test and Mann-Whitney U test. RESULTS: We observed enhanced T cell exhaustion and senescence especially at the tumor site. CD8+ T cells expressed several molecules associated with T cell exhaustion (PD-1, CTLA-4, 2B4, CD160) and T cell senescence (CD57, lack of CD28). This phenotype was associated with lower proliferative capacity and impaired function. Despite a high expression of the transcription factor T-bet, CD8+ T cells from the tumor site failed to produce IFNγ after CD3/CD28 in vitro restimulation and displayed a reduced ability to degranulate in response to T cell stimuli. Notably, the percentage of senescent CD57+CD28- CD8+ T cells was significantly lower in treated myeloma patients when compared to untreated patients. CONCLUSIONS: T cells from the bone marrow of myeloma patients were more severely impaired than peripheral T cells. While our data suggest that terminally differentiated cells are preferentially deleted by therapy, immune-checkpoint molecules were still present on T cells supporting the potential of checkpoint inhibitors to reactivate T cells in myeloma patients in combination therapies. However, additional avenues to restore anti-myeloma T cell responses are urgently needed.

Low Beclin-1 expression predicts improved overall survival in patients treated with immunomodulatory drugs for multiple myeloma and identifies autophagy inhibition as a promising potentially druggable new therapeutic target: an analysis from The Austrian Myeloma Registry (AMR).

Beclin-1 is a key regulator of autophagy and has been suggested to be involved in the development of drug resistance in multiple myeloma (MM). We analyzed the expression of Beclin-1 in a retrospective cohort of 70 MMs. Beclin-1 expression did not influence overall survival (OS) and progression-free survival (PFS) in patients with therapy-naïve MM. In patients treated with immunomodulatory drugs (IMiDs) lack of or low Beclin-1 expression resulted in a significantly improved OS and PFS compared to those treated with bortezomib or nonnovel agents. Beclin-1 expression was more frequently detected in relapsed MM than in therapy-naïve MM probably being a hallmark of tumor progression and therapy resistance. If validated prospectively, Beclin-1 expression might identify patients prone to profit above average from IMiDs and enable a more rational allocation of antimyeloma therapies. Furthermore, the inhibition of autophagy could be a new promising target to improve response to treatment in the relapsed/refractory setting.

CCR10/CCL27 crosstalk contributes to failure of proteasome-inhibitors in multiple myeloma.

The bone marrow microenvironment plays a decisive role in multiple myeloma progression and drug resistance. Chemokines are soluble mediators of cell migration, proliferation and survival and essentially modulate tumor progression and drug resistance. Here we investigated bone marrow-derived chemokines of naive and therapy-refractory myeloma patients and discovered that high levels of the chemokine CCL27, known so far for its role in skin inflammatory processes, correlated with worse overall survival of the patients. In addition, chemokine levels were significantly higher in samples from patients who became refractory to bortezomib at first line treatment compared to resistance at later treatment lines.In vitro as well as in an in vivo model we could show that CCL27 triggers bortezomib-resistance of myeloma cells. This effect was strictly dependent on the expression of the respective receptor, CCR10, on stroma cells and involved the modulation of IL-10 expression, activation of myeloma survival pathways, and modulation of proteasomal activity. Drug resistance could be totally reversed by blocking CCR10 by siRNA as well as blocking IL-10 and its receptor.From our data we suggest that blocking the CCR10/CCL27/IL-10 myeloma-stroma crosstalk is a novel therapeutic target that could be especially relevant in early refractory myeloma patients.

Adipocyte-derived players in hematologic tumors: useful novel targets?

INTRODUCTION: Adipocytes and their products play essential roles in tumor establishment and progression. As the main cellular component of the bone marrow, adipocytes may contribute to the development of hematologic tumors. AREAS COVERED: This review summarizes experimental data on adipocytes and their interaction with various cancer cells. Special focus is set on the interactions of bone marrow adipocytes and normal and transformed cells of the hematopoietic system such as myeloma and leukemia cells. Current in vitro and in vivo data are summarized and the potential of novel therapeutic targets is critically discussed. EXPERT OPINION: Targeting lipid metabolism of cancer cells and adipocytes in combination with standard therapeutics might open novel therapeutic avenues in these cancer entities. Adipocyte-derived products such as free fatty acids and specific adipokines such as adiponectin may be vital anti-cancer targets in hematologic malignancies. However, available data on lipid metabolism is currently mostly referring to peripheral fat cell/cancer cell interactions and results need to be evaluated specifically for the bone marrow niche.