Reena Rani

An update in recurrent spontaneous abortion.

Recurrent spontaneous abortion (RSA) is defined as three or more consecutive pregnancy losses prior to the 20th week of gestation. The etiology of recurrent spontaneous abortion is often unclear and may be multifactorial, with much controversy regarding diagnosis and treatment. Reasonably accepted etiologic causes include, genetics, anatomical, endocrine, placental anomalies, hormonal problems, infection, smoking and alcohol consumption, exposure to environmental factors, psychological trauma and stressful life event, certain coagulation and immunoregulatory protein defects. Detection of an abnormality in any of these areas may result into specific therapeutic measures, with varying degrees of success. However, the majority of cases of RSA remains unexplained and is found to be associated with certain autoimmune (APA, ANA, ACA, ATA, AECA) and alloimmune (APCA, Ab2, MLR-Bf) antibodies that may play major role in the immunologic failure of pregnancy and may lead to abortion. Alteration in the expression of HLA-G molecules, T-helper-1 (Th-1) pattern of cytokines and natural killer (NK) cells activity may also induce abortion. Various forms of treatment like antithrombotic therapies such as aspirin and heparin, intravenous immunoglobulin (IVIg) therapy, immunotherapy with paternal lymphocytes and vitamin D3 therapy are effective mode of treatment for unexplained cause of fetal loss in women with RSA.

Trefoil factor 2 rapidly induces interleukin 33 to promote type 2 immunity during allergic asthma and hookworm infection

The repair protein trefoil factor 2 promotes Th2 responses to helminth infection and allergens in part by inducing IL-33.

TNF-α induces leukemic clonal evolution ex vivo in Fanconi anemia group C murine stem cells

The molecular pathogenesis of the myeloid leukemias that frequently occur in patients with Fanconi anemia (FA) is not well defined. Hematopoietic stem cells bearing inactivating mutations of FA complementation group C (FANCC) are genetically unstable and hypersensitive to apoptotic cytokine cues including IFN-γ and TNF-α, but neoplastic stem cell clones that arise frequently in vivo are resistant to these cytokines. Reasoning that the combination of genetic instability and cytokine hypersensitivity might create an environment supporting the emergence of leukemic stem cells, we tested the leukemia-promoting effects of TNF-α in murine stem cells. TNF-α exposure initially profoundly inhibited the growth of Fancc–/– stem cells. However, longer-term exposure of these cells promoted the outgrowth of cytogenetically abnormal clones that, upon transplantation into congenic WT mice, led to acute myelogenous leukemia. TNF-α induced ROS-dependent genetic instability in Fancc–/– but not in WT cells. The leukemic clones were TNF-α resistant but retained their characteristic hypersensitivity to mitomycin C and exhibited high levels of chromosomal instability. Expression of FANCC cDNA in Fancc–/– stem cells protected them from TNF-α–induced clonal evolution. We conclude that TNF-α exposure creates an environment in which somatically mutated preleukemic stem cell clones are selected and from which unaltered TNF-α–hypersensitive Fancc–/– stem cells are purged.

Oxidative Stress in Fanconi Anemia Hematopoiesis and Disease Progression

Patients with the genomic instability syndrome Fanconi anemia (FA) commonly develop progressive bone marrow failure and have a high risk of cancer. The prominent role of the FA protein family involves DNA damage response and/or repair. Oxidative stress, defined as an imbalance between the production of reactive oxygen species and antioxidant defense, is considered to be an important pathogenic factor in leukemia-prone bone marrow diseases such as FA. Cellular responses inducing resistance to oxidative stress are important for cellular survival, organism lifespan, and cancer prevention, but until recently, mammalian factors regulating resistance to oxidative stress have not been well characterized. Significant evidence supports excessive apoptosis of hematopoietic stem/progenitor cells, induced by stresses, most significantly oxidative stress, as a critical factor in the pathogenesis of bone marrow failure and leukemia progression in FA. In this brief review, we discuss the functional link between FA proteins and oxidative DNA damage response/repair, with emphasis on the implication of oxidative stress in the pathophysiology and abnormal hematopoiesis in FA.

Inflammatory Reactive Oxygen Species-Mediated Hemopoietic Suppression in Fancc-Deficient Mice

Patients with the genomic instability syndrome Fanconi anemia (FA) commonly develop progressive bone marrow (BM) failure and have a high risk of cancer. Certain manifestations of the disease suggest that the FA immune system is dysfunctional and may contribute to the pathogenesis of both BM failure and malignancies. In this study, we have investigated inflammation and innate immunity in FA hemopoietic cells using mice deficient in Fanconi complementation group C gene (Fancc). We demonstrate that Fancc-deficient mice exhibit enhanced inflammatory response and are hypersensitive to LPS-induced septic shock as a result of hemopoietic suppression. This exacerbated inflammatory phenotype is intrinsic to the hemopoietic system and can be corrected by the re-expression of a wild-type FANCC gene, suggesting a potential role of the FANCC protein in innate immunity. LPS-mediated hemopoietic suppression requires two major inflammatory agents, TNF-α and reactive oxygen species. In addition, LPS-induced excessive accumulation of reactive oxygen species in Fancc−/− BM cells overactivates the stress kinase p38 and requires prolonged activation of the JNK. Our data implicate a role of inflammation in pathogenesis of FA and BM failure diseases in general.

TGF-β limits IL-33 production and promotes the resolution of colitis through regulation of macrophage function.

Mϕs promote tissue injury or repair depending on their activation status and the local cytokine milieu. It remains unclear whether the immunosuppressive effects of transforming growth factor β (TGF‐β) serve a nonredundant role in Mϕ function in vivo. We generated Mϕ‐specific transgenic mice that express a truncated TGF‐β receptor II under control of the CD68 promoter (CD68TGF‐βDNRII) and subjected these mice to the dextran sodium sulfate (DSS) model of colitis. CD68TGF‐βDNRII mice have an impaired ability to resolve colitic inflammation as demonstrated by increased lethality, granulocytic inflammation, and delayed goblet cell regeneration compared with transgene negative littermates. CD68TGF‐βDNRII mice produce significantly less IL‐10, but have increased levels of IgE and numbers of IL‐33+ Mϕs than controls. These data are consistent with associations between ulcerative colitis and increased IL‐33 production in humans and suggest that TGF‐β may promote the suppression of intestinal inflammation, at least in part, through direct effects on Mϕ function.

Nucleophosmin regulates cell cycle progression and stress response in hematopoietic stem/progenitor cells.

Nucleophosmin (NPM) is a multifunctional protein frequently overexpressed in actively proliferating cells. Strong evidence indicates that NPM is required for embryonic development and genomic stability. Here we report that NPM enhances the proliferative potential of hematopoietic stem cells (HSCs) and increases their survival upon stress challenge. Both short term liquid culture and clonogenic progenitor cell assays show a selective expansion of NPM-overexpressing HSCs. Interestingly, HSCs infected with NPM retrovirus show significantly reduced commitment to myeloid differentiation compared with vector-transduced cells, and majority of the NPM-overexpressing cells remains primitive during a 5-day culture. Bone marrow transplantation experiments demonstrate that NPM promotes the self-renewal of long term repopulating HSCs while attenuated their commitment to myeloid differentiation. NPM overexpression induces rapid entry of HSCs into the cell cycle and suppresses the expression of several negative cell cycle regulators that are associated with G1-to-S transition. NPM knockdown elevates expression of these negative regulators and exacerbates stress-induced cell cycle arrest. Finally, overexpression of NPM promotes the survival and recovery of HSCs and progenitors after exposure to DNA damage, oxidative stress, and hematopoietic injury both in vivo and in vitro. DNA repair kinetics study suggests that NPM has a role in reducing the susceptibility of chromosomal DNA to damage rather than promoting DNA damage repair. Together, these results indicate that NPM plays an important role in hematopoiesis via mechanisms involving modulation of HSC/progenitor cell cycle progression and stress response.

The FA pathway counteracts oxidative stress through selective protection of antioxidant defense gene promoters

Oxidative stress has been implicated in the pathogenesis of many human diseases including Fanconi anemia (FA), a genetic disorder associated with BM failure and cancer. Here we show that major antioxidant defense genes are down-regulated in FA patients, and that gene down-regulation is selectively associated with increased oxidative DNA damage in the promoters of the antioxidant defense genes. Assessment of promoter activity and DNA damage repair kinetics shows that increased initial damage, rather than a reduced repair rate, contributes to the augmented oxidative DNA damage. Mechanistically, FA proteins act in concert with the chromatin-remodeling factor BRG1 to protect the promoters of antioxidant defense genes from oxidative damage. Specifically, BRG1 binds to the promoters of the antioxidant defense genes at steady state. On challenge with oxidative stress, FA proteins are recruited to promoter DNA, which correlates with significant increase in the binding of BRG1 within promoter regions. In addition, oxidative stress-induced FANCD2 ubiquitination is required for the formation of a FA-BRG1-promoter complex. Taken together, these data identify a role for the FA pathway in cellular antioxidant defense.

Immunological cell type characterization and Th1–Th17 cytokine production in a mouse model of Gaucher disease

Gaucher disease is a lysosomal storage disease resulting from insufficient acid β-glucosidase (glucocerebrosidase, GCase, EC 4.2.1.25) activity and the resultant accumulation of glucosylceramide. Macrophage (Mϕ) lineage cells are thought to be the major disease effectors because of their secretion of numerous cytokines and chemokines that influence other poorly defined immunological cell populations. Increases in several such populations were identified in a Gba1 mouse model (D409V/null; 9V/null) of Gaucher disease including antigen presenting cells (APCs), i.e., Mϕ, dendritic cells (DCs), neutrophils (PMNs), and CD4(+) T cells. FACS analyses showed increases in these cell types in 9V/null liver, spleen lung, and bone marrow. T-cells or APCs enhanced activations were evident by positivity of CD40L, CD69, as well as CD40, CD80, CD86, and MHCII on the respective cells. Mϕ, and, unexpectedly, DCs, PMNs, and T cells, from 9V/null mice showed excess glucosylceramides as potential bases for activation of APCs and T cells to induce Th1 (IFNγ, IL12, TNFα,) and Th17 (IL17A/F) cytokine production. These data imply that excess glucosylceramides in these cells are pivotal for activation of APCs and T cell induction of Th1 and Th17 responses and PMN recruitment in multiple organs of this model of Gaucher disease.

Complement drives glucosylceramide accumulation and tissue inflammation in Gaucher disease

Gaucher disease is caused by mutations in GBA1, which encodes the lysosomal enzyme glucocerebrosidase (GCase). GBA1 mutations drive extensive accumulation of glucosylceramide (GC) in multiple innate and adaptive immune cells in the spleen, liver, lung and bone marrow, often leading to chronic inflammation. The mechanisms that connect excess GC to tissue inflammation remain unknown. Here we show that activation of complement C5a and C5a receptor 1 (C5aR1) controls GC accumulation and the inflammatory response in experimental and clinical Gaucher disease. Marked local and systemic complement activation occurred in GCase-deficient mice or after pharmacological inhibition of GCase and was associated with GC storage, tissue inflammation and proinflammatory cytokine production. Whereas all GCase-inhibited mice died within 4–5 weeks, mice deficient in both GCase and C5aR1, and wild-type mice in which GCase and C5aR were pharmacologically inhibited, were protected from these adverse effects and consequently survived. In mice and humans, GCase deficiency was associated with strong formation of complement-activating GC-specific IgG autoantibodies, leading to complement activation and C5a generation. Subsequent C5aR1 activation controlled UDP-glucose ceramide glucosyltransferase production, thereby tipping the balance between GC formation and degradation. Thus, extensive GC storage induces complement-activating IgG autoantibodies that drive a pathway of C5a generation and C5aR1 activation that fuels a cycle of cellular GC accumulation, innate and adaptive immune cell recruitment and activation in Gaucher disease. As enzyme replacement and substrate reduction therapies are expensive and still associated with inflammation, increased risk of cancer and Parkinson disease, targeting C5aR1 may serve as a treatment option for patients with Gaucher disease and, possibly, other lysosomal storage diseases.