The plethora of immunomodulatory drugs: opportunities for immune-mediated kidney diseases.

In recent decades, insights into the molecular pathways involved in disease have revolutionized the treatment of autoimmune diseases. A plethora of targeted therapies have been identified and are at varying stages of clinical development in renal autoimmunity. Some of these agents, such as rituximab or avacopan, have been approved for the treatment of immune-mediated kidney disease, but kidney disease lags behind more common autoimmune disorders in new drug development. Evidence is accumulating as to the importance of adaptive immunity, including abnormalities in T-cell activation and signaling, and aberrant B-cell function. Furthermore, innate immunity, particularly the complement and myeloid systems, as well as pathologic responses in tissue repair and fibrosis, play a key role in disease. Collectively, these mechanistic studies in innate and adaptive immunity have provided new insights into mechanisms of glomerular injury in immune-mediated kidney diseases. In addition, inflammatory pathways common to several autoimmune conditions exist, suggesting that the repurposing of some existing drugs for the treatment of immune-mediated kidney diseases is a logical strategy. This new understanding challenges the clinical investigator to translate new knowledge into novel therapies leading to better disease outcomes. This review highlights promising immunomodulatory therapies tested for immune-mediated kidney diseases as a primary indication, details current clinical trials and discusses pathways that could be targeted in the future.

Review article: Kidney dendritic cells: their role in homeostasis, inflammation and transplantation.

There is definitive experimental proof that a lattice of dendritic cells (DC) exist within the renal parenchyma. Kidney-resident DC (KDC) are an important constituent of passenger leucocytes that initiate the direct component of allograft rejection in transplantation and form a central element of the innate immune response following injurious stimuli to the kidney. DC are recruited to the kidney in pathophysiological states such as glomerulonephritis and ischaemia-reperfusion injury. However, the exact mechanism for engaging and attracting DC to infectious and transplant antigens, and whether specific DC subsets are involved remains unresolved. In addition, the extent to which resident and infiltrating DC contribute to the propagation of injury or rejection is also unclear. Despite consistently expanding published work regarding DC location, phenotype and function, there are a number of deficiencies in our knowledge base, particularly in relation to KDC.

Endogenous CD100 promotes glomerular injury and macrophage recruitment in experimental crescentic glomerulonephritis.

CD100 participates in adaptive immune responses and is important in neural cell migration. To determine the role of endogenous CD100 in severe glomerular inflammation, we induced experimental crescentic glomerulonephritis by planting a foreign antigen in glomeruli of sensitized normal and CD100-deficient (CD100(-/-)) mice. Fewer CD100(-/-) glomeruli exhibited crescent formation or severe histological changes. Antigen-specific immune responses were reduced in CD100(-/-) mice. There was less interferon (IFN)-gamma and interleukin (IL)-4 production by splenocytes and fewer activated T and B cells were present in lymph nodes of immunized CD100(-/-) mice. Serum antigen-specific immunoglobulin (IgG) levels were also decreased. Glomerular macrophage and CD4(+) cell infiltration, and IgG and C3 deposition were attenuated. Normal kidneys expressed mRNA for CD100 and plexin-B1 (the tissue receptor of CD100). Direct immunofluorescence showed that renal-CD100 protein was predominantly in tubules, while plexin-B1 was present in both glomeruli and tubules. To determine whether glomerular plexin-B1 mediates leucocyte recruitment via leucocyte CD100, recruitment was studied after passive transfer of heterologous antibody (attracting neutrophils) or isologous antibody (attracting macrophages). Glomerular macrophages were reduced in CD100(-/-) mice, but neutrophil recruitment was equivalent, consistent with CD100 expression on macrophages, but not neutrophils. CD100 promotes severe nephritogenic immune responses and leucocyte CD100-glomerular plexin-B1 interactions enhance macrophage recruitment to glomeruli.

Breast cancer metastasis in a human bone NOD/SCID mouse model.

A major dilemma facing patients with breast cancer is how to decide between over treating indolent tumors and failing to adequately treat aggressive, potentially lethal cancers. Determination of the metastatic potential of a patient's breast cancer would clearly help guide those treatment decisions. Breast cancer commonly spreads to bone in 70% of women with advanced disease. However, the mechanism of bone metastasis is not well understood. One possibility is that the microenvironment within bone marrow, highly rich in growth factors and cytokines, is suitable for the proliferation of breast cancer cells. In this study, we developed a method for implanting human bone in NOD/SCID mice and show that the human bone implants are viable for more than 20 weeks. This human bone NOD/SCID mouse model provides an opportunity to functionally characterize human breast cancer cell behavior in an in vivo human microenvironment. Several breast tumor cell lines have been shown to grow in the human-bone-NOD/SCID model system, however each line has a different functional profile. Here we show that cotransplantation of GFP-MDA-MB-231 breast cancer cells with morcellized human bone allows for tissue specific metastasis to an initially tumor free bone implant. Furthermore, metastasis of breast tumor cells to implanted tumor-free human bone was seen when patient bone containing a metastatic breast tumor was implanted in the host mouse. With this model, we can distinguish between primary invasive breast tumors with and without bone metastatic potential.

Veterinary epidemiology: vaccination strategies for foot-and-mouth disease.

When foot-and-mouth disease struck the United Kingdom in 2001, the traditional 'stamping out' policy of 1967-68 was supplemented by the pre-emptive culling of animals in premises contiguous to infected premises. A model proposed by Tildesley et al. indicates that the introduction of vaccination should at least halve the number of premises that would need to be subjected to culling in the event of another outbreak. We contest, however, that the overlapping confidence intervals of the outputs of their model, and the inconsistency of their results compared with those from previous models, call into question the model's value as a decision tool, while adding little to the recognized tenet of ring vaccination.

Expression of TMPRSS2:ERG gene fusion in prostate cancer cells is an important prognostic factor for cancer progression.

The prostate-specific gene, TMPRSS2, is fused with the transcription factor gene, ERG in a high proportion of prostate cancers. However, the clinical significance of TMPRSS2:ERG gene fusion among prostate cancer patients is unknown. We assayed for the presence of the TMPRSS2:ERG gene fusion product among 26 patients who underwent surgery for clinically localized prostate cancer using RT-PCR and direct DNA sequencing, and evaluated its prognostic significance. All 26 patients had cancers of the same histologic grade (Gleason score 7). The fusion protein was present within prostate cancer tumor cells in eleven patients (42.3%). Nine patients experienced biochemical disease relapse (elevated PSA) after a mean follow-up of 12 months (range 1 to 48 months). Patients with the fusion protein had a significantly higher rate of recurrence (5-year recurrence rate 79.5%) compared to patients who lacked the fusion protein (five-year recurrence rate 37.5%, p = 0.009). The adjusted hazard ratio for disease relapse for patients with the fusion protein was 7.1 (95% C.I.: 1.1-45, p = 0.03) compared to patients without the fusion protein. In multivariate analysis, the presence of gene fusion was the single most important prognostic factor. Our study indicates that the expression of TMPRSS2:ERG fusion gene among prostate cancer patients treated with surgery is a strong prognostic factor for disease relapse, and may have important clinical implications.

Novel RING E3 ubiquitin ligases in breast cancer.

Defects in ubiquitin E3 ligases are implicated in the pathogenesis of several human diseases, including cancer, because of their central role in the control of diverse signaling pathways. RING E3 ligases promote the ubiquitination of proteins that are essential to a variety of cellular events. Identification of which ubiquitin ligases specifically affect distinct cellular processes is essential to the development of targeted therapeutics for these diseases. Here we discuss two novel RING E3 ligases, BCA2 and RNF11, that are closely linked to human breast cancer. BCA2 E3 ligase is coregulated with estrogen receptor and plays a role in the regulation of epidermal growth factor receptor (EGF-R) trafficking. RNF11 is a small RING E3 ligase that affects transforming growth factorbeta and EGF-R signaling and is overexpressed in invasive breast cancers. These two proteins demonstrate the complexity of RING E3 ligase interactions in breast cancer and are potential targets for therapeutic interventions.

A novel RING-type ubiquitin ligase breast cancer-associated gene 2 correlates with outcome in invasive breast cancer.

The RING finger family of proteins possess ubiquitin ligase activity and play pivotal roles in protein degradation and receptor-mediated endocytosis. In this study, we examined whether the breast cancer-associated gene 2 (BCA2), a novel RING domain protein, has E3 ubiquitin ligase activity and investigated its expression status in breast tumors. The full-length BCA2 gene was cloned from the human breast cancer cell line MDA-MB-468. It encodes an open reading frame of 304 amino acids and contains a RING-H2 domain. BCA2 maps to chromosome 1q21.1, a region known to harbor cytogenetic aberrations in breast cancers. We found that the BCA2 protein has an intrinsic autoubiquitination activity, the hallmark of E3 ligases, whereas mutant RING protein is not autoubiquitinated. This indicates that the BCA2 ubiquitin ligase activity is dependent on the RING-H2 domain. Using tissue microarrays and immunohistochemistry, we found strong to intermediate BCA2 staining in 56% of 945 invasive breast cancers cases, which was significantly correlated with positive estrogen receptor status [odds ratio (OR), 1.51; P = 0.004], negative lymph node status (OR, 0.73; P = 0.02), and an increase in disease-free survival for regional recurrence (OR, 0.45; P = 0.03). Overexpression of BCA2 increased proliferation and small interfering RNA inhibited growth of T47D human breast cancer cells and NIH3T3 mouse cells. The autoubiquitination activity of BCA2 indicates that it is a novel RING-type E3 ligase. Its association with clinical measures and its effects on cell growth indicate that BCA2 may be important for the ubiquitin modification of proteins crucial to breast carcinogenesis and growth.

Cyclin I is expressed in human breast cancer and closely associated with VEGF and KDR expression.

In the present study, cyclin I protein expression in 114 invasive human breast cancers was correlated with cell cycle and angiogenesis-related proteins and clinico-pathological data. A strong association was found between cytoplasmic cyclin I staining and VEGF (p = 0.001) as well as the VEGF receptor KDR (p = 0.001), suggesting a link between cyclin I and angiogenesis.

The RING finger protein RNF11 is expressed in bone cells during osteogenesis and is regulated by Ets1.

Maturation of MC3T3-E1 osteoblast cells in vitro can be divided into three major stages, namely, proliferation, differentiation, and mineralization. Our microarray analysis identified genes differentially expressed between proliferating and differentiated MC3T3-E1 osteoblastic cells. Immunohistochemical analyses of RNF11 protein encoded by one of the differentially expressed genes revealed it as highly expressed in osteoblasts of multiple skeletal elements during embryonic bone formation in mice. In contrast, cartilage, undifferentiated mesenchymal tissue and osteocytes did not express detectable amounts of the RNF11 protein. The RNF11 mRNA was found to be abundant during the proliferation stage of MC3T3-E1 osteoblast development with a short second peak of expression during the mineralization phase. This pattern of expression is similar to that of the Ets1 transcription factor during osteogenic differentiation of MC3T3-E1 cells, and we used immunohistochemistry to show that, in vivo, Ets1 protein is coexpressed with RNF11 in osteoblasts. The human and mouse RNF11 promoters each contain three Ets transcription factor binding sites (EBS) and we found that Ets1 binds specifically to one of them. The functionality of this site was tested in a transcription-transactivation assay, indicating that RNF11 expression in bone cells is regulated by the Ets1 factor.

Ets2 transcription factor inhibits mineralization and affects target gene expression during osteoblast maturation.

Our goal is to understand how Ets family transcription factors affect the genetic programs that control bone development. Modest overexpression of Ets2 in transgenic mice leads to Down's syndrome-like bone abnormalities. We observed that in the MC3T3-E1 in vitro model of osteoblast development, mature osteoblasts have very high levels of Ets2 relative to the immature preosteoblasts. We hypothesized that overexpression of Ets2 could have noticeable effects on gene expression, and found that exogenous Ets2 expression results in a complete lack of mineralized matrix in stable Ets2 transfected cells. Our cDNA microarray-based expression profiling of preosteoblasts vs. differentiated osteoblasts revealed several genes previously unrecognized as having roles in osteoblast maturation and up-regulated only in the mature osteoblasts. The promoters of these genes and known osteoblast marker genes were examined for Ets transcription factor binding sites (EBSs). Interactions of these sites with Ets2 protein were tested by EMSA. In vitro expressed Ets2 protein was able to form a protein:DNA complex with both known (Bsp, Opn, ON) and novel (Btg2, CysC, Lum) bone-related genes. In addition, Cbfa1 was found to interact with Ets2, forming a complex on the Opn promoter.

The RING-H2 protein RNF11 is differentially expressed in breast tumours and interacts with HECT-type E3 ligases.

A breast cancer-associated mRNA originally cloned as a 475-bp partial cDNA from a library enriched for tumour cDNAs [Oncogene 16 (1998) 327] is expressed at high levels in breast and prostate cancer cells. Immunohistochemical analysis indicates that the protein is expressed in primary breast tumours. We used RT-PCR to generate a full-length 2852 nt mRNA sequence that includes the hypothetical open reading frame (ORF) for human RNF11. Our analysis shows that RNF11 encodes modular domains and motifs likely to interact with other proteins involved in oncogenesis. Chief among these are the RING-H2 finger domain that could facilitate the degradation of specific substrate(s) involved in oncogenesis and the PY motif which binds to WW-domain proteins, several of which are known to be E3 ubiquitin ligases. Our GST-pulldown and immunoprecipitation results indicate that RNF11 interacts with the E3 ligase AIP4 when coexpressed with RNF11 in mammalian cells.

The breast cancer-associated gene Di12 has oncogenic activity.

The breast cancer-associated gene Di12 encodes a novel protein, which was found overexpressed in invasive ductal carcinomas of the breast. In experiments designed to assess the role of the Di12 gene in oncogenesis, the overexpression of 339 N-terminal amino acids of this gene in NIH3T3 cells resulted in cellular transformation and in vivo tumorigenesis. NIH3T3-Di12 tumor cell growth was partly reversible upon Di12 antisense treatment. In addition, transformation of the ER+ human breast cancer cell line MCF-7 resulted in hormone independent growth of these tumors in nude mice. Di12 expression in NIH3T3 and MCF-7 tumor cells was confirmed by RT-PCR and mabDi12 immunostaining. Immunohistochemistry using mabDi12 on an arrayed collection of 106 invasive breast tumors further underlined the expression of the gene in over 75% of advanced stage breast cancers. Our data indicate that Di12 expression is oncogenic in in vitro transformation and in vivo tumorigenic assays.

Gene expression profiling of ductal carcinomas in situ and invasive breast tumors.

UNLABELLED: Comparative and functional genomics are powerful tools to advance the understanding of the molecular basis of cancer. It is believed that genes are epigenetically regulated and, thus, each tumor type and stage will be characterized by a gene expression fingerprint. In this study we identified genes that are differentially expressed in ductal carcinoma in situ and invasive ductal carcinoma of the breast. To isolate genes that are associated with progression of breast cancer we performed differential display and subtractive cloning procedures using matched RNA from normal and tumor tissue. cDNA microarray analysis generated gene expression profiles typical of the transition from in situ to invasive breast cancer when we used mRNA extracted from a case of low- to intermediate-grade DCIS and a case of high-grade DCIS/IDC. cDNAs from these samples were the probes in a cDNA microarray hybridization to 9183 unique cDNAs representing 8507 genes. Signals from both transcriptomes were obtained for 8083 genes, and the balanced differential expression values between pure DCIS and DCIS/invasive tumors revealed 303 distinct cDNAs with a ratio of > 2. Interferon inducible genes were found to be expressed at the highest level in the pure DCIS sample. Genes most abundantly expressed in the invasive tumor were immunoglobulin heavy constant gamma 3 and calgranulin B. Further analysis of RNA and protein expression in breast tumor cell lines and patient tissue samples revealed that: IGFBP-rP1 is down-regulated in invasive tumors whereas cyclin I protein is regulated by ubiquitination and is associated with ER-negative breast cancers. CONCLUSION: The known and novel genes discussed here represent targets for molecular characterization during breast cancer development as well as for designing novel strategies for diagnosis and treatment.

Characterization of a novel human breast cancer associated gene (BCA3) encoding an alternatively spliced proline-rich protein.

As part of an integrated study of breast cancer gene expression, partial cDNAs were cloned from normal and tumor breast cells by subtractive-hybridization and differential display cloning. The DNA sequence for one of these breast cancer associated genes was used to construct the larger 1319 bp BCA3 cDNA sequence using ESTs without assigned names or functions. High-level BCA3 mRNA expression was found in breast and prostate tumor cell lines whereas normal breast and prostate tissues have low-level expression. Further analysis revealed possible functional domains and alternative splicing of BCA3 that we confirmed by RT-PCR analysis. Immunohistochemistry revealed that the protein is expressed in breast tumor cells in vivo, and not in surrounding stromal tissue.

Coordinate gene expression patterns during osteoblast maturation and retinoic acid treatment of MC3T3-E1 cells.

The clonal osteoblast-like cell line MC3T3-E1 undergoes time-dependent morphological changes leading to the formation of bone nodules in vitro. Serial analysis of gene expression was used to identify genes expressed in MC3T3-E1 cells, including known osteoblast markers, structural and matrix proteins, transcription factors, cell-cycle regulators, and housekeeping genes. Relative changes in the expression of 92 representative transcripts were determined by arrayed cDNA hybridization. Complex probes were derived from MC3T3-E1 cells during the proliferation, differentiation, and matrix mineralization stages, and from cells grown with all- trans-retinoic acid (RA), a potent bone morphogen. We found that the relative expression of 68 of these genes was higher during differentiation than in the earlier proliferative phase. In the mineralization phase, all but 16 cDNAs had lower normalized hybridization intensity ratios as compared with the differentiation phase. cDNAs for vimentin (Vim), presenilin 2 (Psen2), guanine nucleotide binding protein alpha stimulating (Gnas), gap-junction membrane channel protein beta 1 (Gjb1), fibroblast growth factor receptor 1 (Fgfr1), eukaryotic translation elongation factor 2 (Eef2), and calponin 2 (Cnn2) had higher normalized hybridization intensities in both differentiation and mineralization than in proliferation. RA treatment during the differentiation phase appeared to reduce the expression of the 92 genes examined, as 62 cDNAs had lower hybridization intensities with complex cDNA probes derived from RA-treated cells than with the probe from untreated cells. Several cDNAs representing genes with previously unrecognized RA responsiveness were identified by this comparison, including the receptor for bone morphogenetic proteins 2 and 4 (Bmp2/Bmp4) and hemoglobin Y.