Pattern of biopsy-proven renal disease in Sabah: A retrospective cross-sectional study over 3.5 years.

OBJECTIVES: To explore the epidemiological and histopathological patterns of glomerular diseases in Sabah. METHODS: A state-wide cross-sectional study was conducted. There were 336 native renal biopsies in 296 eligible patients from 1st January 2013 to 30th June 2016. All patients aged ≥12 years with sufficient sampling (≥8 glomeruli) for histopathological assessment were included. Graft kidney biopsies, protocol-based biopsies and patients with uncertain demographics were excluded. Demographics of patients, clinical data, laboratory parameters prior to biopsy, and histology findings of renal biopsies were collected from local unit database and recorded into a standardised data collection form. Descriptive statistical analyses were employed and factors associated with Lupus nephritis (LN) were explored using logistic regression. RESULTS: The mean age during biopsy was 34.53 years (Standard Deviation 0.759). Primary glomerulonephritis (PGN) accounted for 42.6% (126) of all native renal biopsies. The commonest cause of PGN was minimal change disease (38.9%, 49) followed by focal segmental glomerulosclerosis (33.3%, 42) and IgA nephropathy (14.3%, 18). LN is the leading cause for secondary glomerulonephritis (SGN) (87.2%, 136). Younger age (Odds Ratio, OR 0.978; 95% Confidence Interval, 95%CI 0.960, 0.996); female gender (OR 17.53; p<0.001); significant proteinuria (OR 132.0; p<0.001); creatinine level at biopsy (OR 11.26; p=0.004); positive antinuclear antibody (ANA) (OR 46.7; p<0.001); and ANA patterns (OR 8.038; p=0.018) were significant in predicting the odds of having LN. CONCLUSION: This is the first epidemiology study of glomerular diseases in Sabah. The predominance of LN suggests lower threshold for renal biopsy in patients with suspected glomerular disorders. We have identified significant predictors for early detection and treatment of LN.

Effect of algae or fish oil supplementation and porcine maternal stress on the adrenal transcriptome of male offspring fed a low-quality protein diet.

Offspring adrenal function may be negatively affected in utero by maternal stressors such as microbial infection. Maternal supplementation with immunomodulatory compounds such as omega-3 polyunsaturated fatty acids (n-3 PUFA) may help minimize the adverse effects of maternal stress on fetal hypothalamic-pituitary-adrenal development and improve offspring health. Presently, n-3 PUFA sources are primarily fish-based, but n-3 PUFA microalgae (AL) may be an alternative. Previously, it was determined that maternal AL or fish oil (FO) supplementation to sows, in addition to maternal stress induced by Escherichia coli lipopolysaccharide (LPS) challenge appeared to have a greater influence on the stress response of male offspring compared to females. To further elaborate on these findings, this study assessed the effects of maternal AL or FO supplementation combined with a maternal LPS challenge on adrenal gene expression in male offspring fed a nursery diet containing low-quality protein sources. Forty-eight sows were fed gestation diets starting on gestation day (gd) 75 containing either 3.12% AL, 3.1% FO, or a control diet containing 1.89% corn oil. On gd 112, half the sows in each treatment were administered 10 â€‹µg/kg LPS i.m. Piglets were weaned at 21 days of age onto a common low-quality plant-based protein diet, and one week after weaning, four piglets per sow were administered 40 â€‹µg/kg LPS i.m. Two hours later, the piglets were euthanized to obtain adrenal tissue, and total RNA was extracted to carry out transcriptome analysis using the Affymetrix GeneChip WT Plus assay and subsequent validation by real-time PCR. Analysis revealed that adrenal steroidogenesis, fatty acid metabolism and immune function were significantly influenced by maternal diet and stress. Increased expression of immune-related genes including lymphocyte antigen 96, TLR-2 and NF-κB suggests that maternal AL supplementation may increase offspring sensitivity to inflammation after weaning. Decreased expression of lymphocyte antigen 96 in male offspring from sows receiving maternal LPS challenge also suggests a possible role of maternal stress in diminishing the offspring immune response to immune stress challenge. Increased expression of the genes encoding the 11BHSD2 enzyme in offspring from sows fed FO may also reduce the magnitude of the stress response. These data provide insight to the immune and metabolic mechanisms that may be influenced by maternal diet and stress.

Microalgae supplementation to late gestation sows and its effects on the health status of weaned piglets fed diets containing high- or low-quality protein sources.

Maternal stress, such as a bacterial infection occurring in late gestation, may predispose offspring to a variety of diseases later in life. It may also alter programming of developing systems within the fetus, such as the hypothalamic-pituitary-adrenal (HPA) axis and immune system. Dietary supplementation during the last trimester of pregnancy with immune-modulating compounds may be a means of reducing potential adverse effects of maternal stress on the developing fetus. Essential omega-3 polyunsaturated fatty acids (n-3 PUFA) such as docosahexanoic acid (DHA) and eicosapentanoic acid are well-known for their immune-modulating and anti-inflammatory properties. Sources of these n-3 PUFA include fish products such as fish oil and microalgae, which may be a suitable alternative to fish-based products. The aim of this study was to determine the effect of supplementing gestating sow diets with n-3 PUFA and inducing an immune stress challenge in late gestation on piglet growth and immune responsiveness when placed on either a high- or low-quality protein diet after weaning. Forty-eight sows were fed gestation diets containing either 3.12% microalgae, 3.1% fish oil or a corn oil control diet containing 1.89% corn oil starting on gestation day (gd) 75. On gd112, half the sows in each treatment were immune stress challenged with bacterial lipopolysaccharide (LPS) endotoxin (10 µg/kg administered i.m). After farrowing, piglet BW gain was monitored weekly during lactation and pigs were weaned at 21 days of age. One week after weaning, four piglets per sow were immune stress challenged with LPS (40 µg/kg administered i.m.). At the same time, four piglets per sow were vaccinated with the novel antigens chicken ovalbumin (OVA) and Candida cellular antigen (CAA) and received booster vaccinations two weeks later. Four weeks after the initial vaccination, a transdermal hypersensitivity immune challenge was performed using the same antigens. Blood samples were also collected to quantify IgG antibody responses to both antigens. PUFA enrichment in sow blood and piglet brain was detected after sows were on feed for 40 days. Piglet growth was increased in pigs fed a high-quality diet in nursery phase 1. Concentrations of the cytokines IL-1ra, IL-6 and IL-10 were elevated in pigs fed a high-quality protein diet following LPS immune challenge. Overall, it appears that in the current study piglet nursery diet quality was more important for determining piglet health and growth than maternal diet and immune stress.

Loss of function of NF1 is a mechanism of acquired resistance to endocrine therapy in lobular breast cancer.

Background: Invasive lobular carcinoma (ILC) as a disease entity distinct from invasive ductal carcinoma (IDC) has merited focused studies of the genomic landscape, but those to date are largely limited to the assessment of early-stage cancers. Given that genomic alterations develop as acquired resistance to endocrine therapy, studies on refractory ILC are needed. Patients and methods: Tissue from 336 primary-enriched, breast-biopsied ILC and 485 estrogen receptor (ER)-positive IDC and metastatic biopsy specimens from 180 ILC and 191 ER-positive IDC patients was assayed with hybrid-capture-based comprehensive genomic profiling for short variant, indel, copy number variants, and rearrangements in up to 395 cancer-related genes. Results: Whereas ESR1 alterations are enriched in the metastases of both ILC and IDC compared with breast specimens, NF1 alterations are enriched only in ILC metastases (mILC). NF1 alterations are predominantly under loss of heterozygosity (11/14, 79%), are mutually exclusive with ESR1 mutations [odds ratio = 0.24, P < 0.027] and are frequently polyclonal in ctDNA assays. Assessment of paired specimens shows that NF1 alterations arise in the setting of acquired resistance. An in vitro model of CDH1 mutated ER-positive breast cancer demonstrates that NF1 knockdown confers a growth advantage in the presence of 4-hydroxy tamoxifen. Our study further identified a significant increase in tumor mutational burden (TMB) in mILCs relative to breast ILCs or metastatic IDCs (8.9% >20 mutations/mb; P < 0.001). Most TMB-high mILCs harbor an APOBEC trinucleotide signature (14/16; 88%). Conclusions: This study identifies alteration of NF1 as enriched specifically in mILC. Mutual exclusivity with ESR1 alterations, polyclonality in relapsed ctDNA, and de novo acquisition suggest a role for NF1 loss in endocrine therapy resistance. Since NF1 loss leads to RAS/RAF kinase activation, patients may benefit from a matched inhibitor. Moreover, for an independent subset of mILC, TMB was elevated relative to breast ILC, suggesting possible benefit from immune checkpoint inhibitors.

Recurrent hyperactive ESR1 fusion proteins in endocrine therapy-resistant breast cancer.

Background: Estrogen receptor-positive (ER-positive) metastatic breast cancer is often intractable due to endocrine therapy resistance. Although ESR1 promoter switching events have been associated with endocrine-therapy resistance, recurrent ESR1 fusion proteins have yet to be identified in advanced breast cancer. Patients and methods: To identify genomic structural rearrangements (REs) including gene fusions in acquired resistance, we undertook a multimodal sequencing effort in three breast cancer patient cohorts: (i) mate-pair and/or RNAseq in 6 patient-matched primary-metastatic tumors and 51 metastases, (ii) high coverage (>500×) comprehensive genomic profiling of 287-395 cancer-related genes across 9542 solid tumors (5216 from metastatic disease), and (iii) ultra-high coverage (>5000×) genomic profiling of 62 cancer-related genes in 254 ctDNA samples. In addition to traditional gene fusion detection methods (i.e. discordant reads, split reads), ESR1 REs were detected from targeted sequencing data by applying a novel algorithm (copyshift) that identifies major copy number shifts at rearrangement hotspots. Results: We identify 88 ESR1 REs across 83 unique patients with direct confirmation of 9 ESR1 fusion proteins (including 2 via immunoblot). ESR1 REs are highly enriched in ER-positive, metastatic disease and co-occur with known ESR1 missense alterations, suggestive of polyclonal resistance. Importantly, all fusions result from a breakpoint in or near ESR1 intron 6 and therefore lack an intact ligand binding domain (LBD). In vitro characterization of three fusions reveals ligand-independence and hyperactivity dependent upon the 3' partner gene. Our lower-bound estimate of ESR1 fusions is at least 1% of metastatic solid breast cancers, the prevalence in ctDNA is at least 10× enriched. We postulate this enrichment may represent secondary resistance to more aggressive endocrine therapies applied to patients with ESR1 LBD missense alterations. Conclusions: Collectively, these data indicate that N-terminal ESR1 fusions involving exons 6-7 are a recurrent driver of endocrine therapy resistance and are impervious to ER-targeted therapies.

FOXC1 regulates the functions of human basal-like breast cancer cells by activating NF-κB signaling.

Human basal-like breast cancer (BLBC) is an enigmatic and aggressive malignancy with a poor prognosis. There is an urgent need to identify therapeutic targets for BLBC, because current treatment modalities are limited and not effective. The forkhead box transcription factor FOXC1 has recently been identified as a critical functional biomarker for BLBC. However, how it orchestrates BLBC cells was not clear. Here we show that FOXC1 activates the transcription factor nuclear factor-κB (NF-κB) in BLBC cells by increasing p65/RelA protein stability. High NF-κB activity has been associated with estrogen receptor-negative breast cancer, particularly BLBC. The effect of FOXC1 on p65/RelA protein stability is mediated by increased expression of Pin1, a peptidyl-prolyl isomerase. FOXC1 requires NF-κB for its regulation of cell proliferation, migration and invasion. Notably, FOXC1 overexpression renders breast cancer cells more susceptible to pharmacological inhibition of NF-κB. These results suggest that BLBC cells may rely on FOXC1-driven NF-κB signaling. Interventions of this pathway may provide modalities for the treatment of BLBC.

A SNP in steroid receptor coactivator-1 disrupts a GSK3ß phosphorylation site and is associated with altered tamoxifen response in bone.

The coregulator steroid receptor coactivator (SRC)-1 increases transcriptional activity of the estrogen receptor (ER) in a number of tissues including bone. Mice deficient in SRC-1 are osteopenic and display skeletal resistance to estrogen treatment. SRC-1 is also known to modulate effects of selective ER modulators like tamoxifen. We hypothesized that single nucleotide polymorphisms (SNP) in SRC-1 may impact estrogen and/or tamoxifen action. Because the only nonsynonymous SNP in SRC-1 (rs1804645; P1272S) is located in an activation domain, it was examined for effects on estrogen and tamoxifen action. SRC-1 P1272S showed a decreased ability to coactivate ER compared with wild-type SRC-1 in multiple cell lines. Paradoxically, SRC-1 P1272S had an increased protein half-life. The Pro to Ser change disrupts a putative glycogen synthase 3 (GSK3)ß phosphorylation site that was confirmed by in vitro kinase assays. Finally, knockdown of GSK3ß increased SRC-1 protein levels, mimicking the loss of phosphorylation at P1272S. These findings are similar to the GSK3ß-mediated phospho-ubiquitin clock previously described for the related coregulator SRC-3. To assess the potential clinical significance of this SNP, we examined whether there was an association between SRC-1 P1272S and selective ER modulators response in bone. SRC-1 P1272S was associated with a decrease in hip and lumbar bone mineral density in women receiving tamoxifen treatment, supporting our in vitro findings for decreased ER coactivation. In summary, we have identified a functional genetic variant of SRC-1 with decreased activity, resulting, at least in part, from the loss of a GSK3ß phosphorylation site, which was also associated with decreased bone mineral density in tamoxifen-treated women.

Nosocomial transmission of parainfluenza 3 virus in hematological patients characterized by molecular epidemiology.

We report an outbreak of parainfluenza 3 virus involving 17 hematology-oncology patients on 2 hospital wards. Sequence analysis of clinical samples confirmed homology of strains for 16/17 patients and 1 healthcare worker. Epidemiological analysis of the outbreak supported the molecular data with nosocomial transmission of the same genetic strain as the cause of the outbreak.

Proteasome inhibition represses ERalpha gene expression in ER+ cells: a new link between proteasome activity and estrogen signaling in breast cancer.

Estrogen receptor-alpha (ERalpha) is a major therapeutic target of hormonal therapies in breast cancer, and its expression in tumors is predictive of clinical response. Protein levels of ERalpha are tightly controlled by the 26S proteasome; yet, how the clinical proteasome inhibitor, bortezomib, affects ERalpha regulation has not been studied. Bortezomib selectively inhibits the chymotrypsin-like activity of the proteasome. Unlike other laboratory proteasome inhibitors, bortezomib failed to stabilize ERalpha protein at a dose exceeding 90% inhibition of the chymotrypsin-like activity. Unexpectedly, however, chronic bortezomib exposure caused a reduction of ERalpha levels in multiple ER+ breast cancer cell lines. This response can be explained by the fact that bortezomib induced a dramatic decrease in ERalpha mRNA because of direct transcriptional inhibition and loss of RNA polymerase II recruitment on the ERalpha gene promoter. Bortezomib treatment resulted in promoter-specific changes in estrogen-induced gene transcription that related with occupancy of ERalpha and RNA polymerase II (PolII) on endogenous promoters. In addition, bortezomib inhibited estrogen-dependent growth in soft agar. These results reveal a novel link between proteasome activity and expression of ERalpha in breast cancer and uncover distinct roles of the chymotrypsin-like activity of the proteasome in the regulation of the ERalpha pathway.

Developmental and hormonal signals dramatically alter the localization and abundance of insulin receptor substrate proteins in the mammary gland.

Insulin receptor substrates (IRS) are central integrators of hormone, cytokine, and growth factor signaling. IRS proteins can be phosphorylated by a number of signaling pathways critical to normal mammary gland development. Studies in transgenic mice that overexpress IGF-I in the mammary gland suggested that IRS expression is important in the regulation of normal postlactational mammary involution. The goal of these studies was to examine IRS expression in the mouse mammary gland and determine the importance of IRS-1 to mammary development in the virgin mouse. IRS-1 and -2 show distinct patterns of protein expression in the virgin mouse mammary gland, and protein abundance is dramatically increased during pregnancy and lactation, but rapidly lost during involution. Consistent with hormone regulation, IRS-1 protein levels are reduced by ovariectomy, induced by combined treatment with estrogen and progesterone, and vary considerably throughout the estrous cycle. These changes occur without similar changes in mRNA levels, suggesting posttranscriptional control. Mammary glands from IRS-1 null mice have smaller fat pads than wild-type controls, but this reduction is proportional to the overall reduction in body size. Development of the mammary duct (terminal endbuds and branch points) is not altered by the loss of IRS-1, and pregnancy-induced proliferation is not changed. These data indicate that IRS undergo complex developmental and hormonal regulation in the mammary gland, and that IRS-1 is more likely to regulate mammary function in lactating mice than in virgin or pregnant mice.

Rapid induction of IGF-IR signaling in normal and tumor tissue following intravenous injection of IGF-I in mice.

The detection of IGF-IR signaling in animal models has important implications for determining the role of this receptor in normal physiology and tumor growth. While many reports have correlated changes in plasma IGF-I levels in vivo with biological responses, few have shown that altered IGF-I levels can directly affect signaling within normal or tumor tissue. Here, we present new data that shows how the intravenous (IV) injection of IGF-I can be used to directly examine IGF signaling at the tissue level. Tail-vein IV injection of IGF-I into mice resulted in a rapid and dose-dependent activation of the IGF-I receptor and downstream phosphorylation of Akt and ERK1/2 in liver, kidney, and mammary gland. Similarly, IV IGF-I rapidly stimulated signaling in HT-29 colorectal and in MCF-7 breast cancer xenografts. This study shows how IV IGF injection can be used to examine the signaling mechanisms used by IGF-IR, in both normal mammary tissue and during tumor growth, and may provide a model for the characterization of IGF inhibitors.

Genetic manipulation of the IGF-I axis to regulate mammary gland development and function.

Insulin-like growth factor I (IGF-I) is known to regulate mammary gland development. This regulation occurs through effects on both cell cycle progression and apoptosis. Our laboratory has studied the IGF-I-dependent regulation of these processes by using transgenic and knockout mouse models that exhibit alterations in the IGF-I axis. Our studies of transgenic mice that overexpress IGF-I during pregnancy and lactation have demonstrated that this growth factor slows the apoptotic loss of mammary epithelial cells during the declining phase of lactation but has minimal effects during early lactation on milk composition or lactational capacity. In contrast, our analysis of early developmental processes in mammary tissue from mice carrying a targeted mutation in the IGF-I receptor gene suggests that IGF-dependent stimulation of cell cycle progression is more important to early mammary gland development than potential anti-apoptotic effects. With both models, the effects of perturbing the IGF-I axis are dependent on the physiological state of the animal. The diminished ductal development that occurs in response to loss of the IGF-I receptor is dramatically restored during pregnancy, whereas the ability of overexpressed IGF-I to protect mammary cells from apoptosis does not occur if the mammary gland is induced to undergo forced involution. Data from our laboratory on the expression of IGF-signaling molecules in the mammary gland suggest that this effect of physiological context may be related to the expression of members of the insulin receptor substrate family.

Growth and characterization of N-methyl-N-nitrosourea-induced mammary tumors in intact and ovariectomized rats.

It is well established that 85-90% of chemically induced mammary tumors in rats will disappear or diminish significantly in size after the ovaries are removed from the animal. However, it is less well established whether a high percentage of these mammary tumors will grow back with prolonged time after ovariectomy. It is also not known what changes in gene expression take place in the tumors as they develop an independence from hormones for growth. This study was carried out to investigate this. Virgin, 50-day-old female Sprague-Dawley rats were injected with N-methyl-N-nitrosourea (MNU) at the dose of 50 mg MNU/kg body wt. When at least one mammary tumor had grown to 1.0-1.5 cm in one dimension, the animal was bilaterally ovariectomized and reduction and then re-growth of the tumors monitored. Control animals were treated identically except they were not ovariectomized when tumors appeared. Re-growths and new tumors and tumors that developed in the control rats were removed when they reached 1.0-1.5 cm in diameter and all animals were killed 25 weeks after the MNU injection. All the animals in the study (100%) developed mammary tumors after MNU injection with an average latency of 56.5 days. After ovariectomy, 93% of the tumors showed 50% or more reduction in size and 76% of the tumors could not be detected by palpation. However, in 96% of the animals where tumor reduction or disappearance occurred, a re-growth or new mammary tumor development took place with an average latency period of 52.8 days from the day of ovariectomy. Of these post-ovariectomy tumors, 36% occurred at a location where tumors had developed prior to ovariectomy, but 64% appeared at new locations. The circulating levels of 17beta-estradiol (E2) was undetectable in the ovariectomized (OVX) rats and significant reduction was seen in the serum concentrations of progesterone (P4), prolactin (PRL), growth hormone (GH) and insulin-like growth factor-I (IGF-I). The tumors from the OVX rats showed indications of progression as evident from loss of differentiation and invasive characteristics. Comparison between tumors from OVX and intact rats revealed a significantly increased expression of P450 aromatase and elevated activation of extracellular signal-regulated kinase 1 and 2, but reduced levels of the progesterone receptor and cyclin D1 in OVX rats. However, the estrogen receptor (ER) content remained similar in tumors from both groups, at least at the protein level, and so did the expression of IGF-I, IGF-II, insulin receptor substrate-1 (IRS1), IRS-2 and epidermal growth factor receptor. IGF-I receptor (IGF-IR) and ErbB-2 were expressed, respectively, in 50 and 70% of the tumors from the OVX animals, whereas these genes were expressed in 100% of the tumors from the intact rats. It is concluded that chemically induced rat mammary tumors may still depend on the ER and local syntheses of E2 and growth factors for growth initially after ovariectomy. However, as these tumors progress, they develop a more aggressive phenotype and lose their dependency on the ER and possibly growth factors.