Novel urinary biomarkers and their association with urinary heavy metals in chronic kidney disease of unknown aetiology in Sri Lanka: a pilot study

Introduction: Chronic kidney disease of unknown etiology (CKDu) has emerged as a significant public health problem in Sri Lanka. The role of environmental exposure to cadmium and arsenic in the aetiology of CKDu is still unclear. Identification of a panel of novel urinary biomarkers would be invaluable in the study of toxin mediated damage postulated to be the aetiology of CKDu. Objectives: The aims of this study were to evaluate the profile of novel urinary biomarkers in CKDu patients and identify any association with environmental exposure to heavy metals. Methods: Thirty seven randomly selected CKDu patients attending a renal clinic in the North Central Province and two control groups namely a farmer group (n=39) and a non-farmer group (n=40) from a non-endemic area were included in this comparative cross sectional study. Urine samples were analyzed for heavy metals and five urinary biomarkers. Results: CKDu patients had significantly elevated urinary levels of fibrinogen (198.2 ng/mg creatinine p<0.001), clusterin (3479 ng/mg creatinine p<0.001), cystatin-C (5124.8 ng/mg creatinine p<0.001) and ß2-microglobulin (9913.4 ng/mg creatinine p<0.001) compared to the control groups. Fibrinogen and ß2-microglobulin were the best to discriminate CKDu patients from normal individuals with the receiver operator areas under the curve being 0.867 and 0.853, respectively. Urinary fibrinogen and KIM-1 levels correlated positively with urinary arsenic levels. KIM-1 levels correlated positively with urinary mercury and lead levels but no correlation was seen with urinary cadmium levels. Conclusions: Fibrinogen and ß2-microglobulin have the potential of being a screening tool for detection of CKDu and may aid the early diagnosis of toxin mediated tubular injury in CKDu. Their usefulness need to be further validated in a larger epidemiological study of patients with early stages of CKDu.

Validation of wet mount microscopy against Trichomonas culture among women of reproductive age group in Western province, Sri Lanka.

Wet mount microscopy is the most commonly used diagnostic method for trichomoniasis in clinical diagnostic services all over the world including Sri Lanka due to its availability, simplicity and is relatively inexpensive. However, Trichomonas culture and PCR are the gold standard tests. Unfortunately, neither the culture nor PCR is available for the diagnosis of trichomoniasis in Sri Lanka. Thus, it is important to validate the wet mount microscopy as it is the only available diagnostic test and has not been validated to date in Sri Lanka. The objective was to evaluate the validity and reliability of wet mount microscopy against gold standard Trichomonas culture among clinic based population of reproductive age group women in Western province, Sri Lanka. Women attending hospital and institutional based clinics were enrolled. They were interviewed and high vaginal swabs were taken for laboratory diagnosis by culture and wet mount microscopy. There were 601 participants in the age group of 15-45 years. Wet mount microscopy showed 68% sensitivity, 100% specificity, 100% positive (PPV) and 98% negative predictive values (NPV) (P=0.001, kappa=0.803) respectively against the gold standard culture. The area under the ROC curve was 0.840. Sensitivity of wet mount microscopy is low. However it has high validity and reliability as a specific diagnostic test for trichomoniasis. If it is to be used among women of reproductive age group in Western province, Sri Lanka, a culture method could be adopted as a second test to confirm the negative wet mount for symptomatic patients.

Use of culture and immunochromatographic technique for diagnosis of trichomoniasis in Sri Lanka.

As a majority of the trichomoniasis patients are asymptomatic, laboratory tests are crucial in case detection. The usefulness of culture and immunochromatographic technique (ICT) compared to microscopy for detection of trichomoniasis in Sri Lanka was assessed. Females (16-45 years) from Colombo district were screened for Trichomonas vaginalis using three laboratory tests namely, microscopy of wet smear, Trichomonas liquid culture and ICT (OSOM® trichomonas rapid test). Trichomoniasis by at least one test being positive was 4.8%. Microscopy, culture and ICT detected 2.8%, 4.2% and 10% cases respectively. Microscopy missed 32% of culture positives. ICT is a simple, practical and reliable alternative to microscopy in laboratory diagnosis of trichomoniasis.

p53 and Notch signaling in chronic lymphocytic leukemia: clues to identifying novel therapeutic strategies.

The p53 tumor suppressor protein has a key role in the induction of apoptosis of chronic lymphocytic leukemia (CLL) cells. Abnormalities within the p53 pathway identify a subset of patients with a poor prognosis. This review describes recent advances in understanding the mechanisms that regulate p53 levels and the role of p53 in the control of the cell cycle and of apoptosis. The classical model of p53-mediated apoptosis emphasizes the transcriptional activation of proapoptotic genes. In contrast, a novel model emphasizes p53's non-transcriptional actions as the major route of apoptosis induction, whereas its transcriptional arm predominantly upregulates antiapoptotic genes, thus providing a negative feedback mechanism that limits apoptosis. Further studies have identified the Notch pathway as a candidate p53-induced antiapoptotic mechanism. In contrast to the classical model, the novel model predicts that pharmacological inhibition of p53's transcriptional function or of the Notch signaling pathway will augment apoptosis induction by cytotoxic agents. Therapeutic strategies based on the novel model, which we review here for the first time, may significantly augment the antitumor actions of cytotoxic agents in CLL and in other malignancies.

Aberrantly activated anti-apoptotic signalling mechanisms in chronic lymphocytic leukaemia cells: clues to the identification of novel therapeutic targets.

Chronic lymphocytic leukaemia (CLL) is the commonest haematological malignancy in the western world and is incurable by cytotoxic therapy. Considerable research effort has identified the signal transduction pathways in CLL cells that contribute to anti-apoptotic signalling. Some pathways are constitutively activated in CLL cells but upregulated in normal cells only when protein tyrosine kinases (PTKs) are activated by ligands. This review describes which PTKs are aberrantly activated in CLL cells and are potential targets for inhibition. Additional potential targets within pathways downstream of these PTKs include Mek/Erk, mTorc1, protein kinase C, PI-3 kinase/Akt, nuclear factor-κB and cyclin-dependent protein kinase. Numerous studies have identified chemical agents and antibodies that selectively kill CLL cells, irrespective of their genetic resistance to conventional chemotherapeutic agents, and which can overcome cytoprotective microenvironmental signalling. These studies have resulted in identification of novel therapies, some of which are currently undergoing clinical trials. In vitro and animal model studies and clinical trials could determine which inhibitors of which targets are the likely to be most effective and least toxic either singly or in combination.

Differential cytopathology and kinetics of measles oncolysis in two primary B-cell malignancies provides mechanistic insights.

Clinical trials using vaccine measles virus (MV) as anticancer therapy are already underway. We compared the oncolytic potential of MV in two B-cell malignancies; adult acute lymphoblastic leukemia (ALL, an aggressive leukemia) and chronic lymphocytic leukemia (CLL, an indolent leukemia overexpressing Bcl-2) using patient-derived material. In vitro, distinct cytopathological effects were observed between MV-infected primary ALL and CLL cells, with large multinucleated syncytia forming in ALL cultures compared to minimal cell-to-cell fusion in infected CLL cells. Cell viability and immunoblotting studies confirmed rapid cell death in MV-infected ALL cultures and slower MV oncolysis of CLL cells. In cell lines, overexpression of Bcl-2 diminished MV-induced cell death providing a possible mechanism for the slower kinetic of MV oncolysis in CLL. In vivo, intratumoral MV treatment of established subcutaneous ALL xenografts had striking antitumor activity leading to complete resolution of all tumors. The antitumor activity of MV was also evident in disseminated ALL xenograft models. In summary, both ALL and CLL are targets for MV-mediated lysis albeit with different kinetics. The marked sensitivity of both primary ALL cells and ALL xenografts to MV oncolysis highlights the tremendous potential of MV as a novel replicating-virus therapy for adult ALL.

The JAK3-selective inhibitor PF-956980 reverses the resistance to cytotoxic agents induced by interleukin-4 treatment of chronic lymphocytic leukemia cells: potential for reversal of cytoprotection by the microenvironment.

Extensive evidence suggests that the malignant cells of chronic lymphocytic leukemia (CLL) patients are in close contact with activated T lymphocytes, which secrete a range of cytoprotective cytokines including interleukin-4 (IL-4). IL-4 induced the rapid phosphorylation and activation of the signal transducer and activator of transcription 6 transcription factor in CLL cells in vitro. Longer incubation with IL-4 resulted in up-regulation of the antiapoptotic proteins, Mcl-1 and Bcl-X(L). All of these events were blocked by the JAK3-selective inhibitor, PF-956980. A dye reduction cytotoxicity assay showed that IL-4 induced resistance to the cytotoxic drugs fludarabine and chlorambucil and to the novel p53-elevating agent nutlin 3. IL-4-induced drug resistance was reversed by PF-956980. These conclusions were confirmed by independent assays for apoptosis induction (annexin V binding, cleavage of poly[ADP-ribose] polymerase, and morphologic analysis). Coculture with bone marrow stromal cells in the presence of supernatants derived from activated T-lymphocyte cultures also protected CLL cells from apoptosis induction by chlorambucil. Protection by these combined signals was reversed by PF-956980. The data here provide a preclinical rationale for the possible therapeutic use of PF-956980 in conjunction with conventional cytotoxic drugs to achieve more extensive killing of CLL cells by overcoming antiapoptotic signaling by the microenvironment.

Common variants at the GCK, GCKR, G6PC2-ABCB11 and MTNR1B loci are associated with fasting glucose in two Asian populations.

AIMS/HYPOTHESIS: To test fasting glucose association at four loci recently identified or verified by genome-wide association (GWA) studies of European populations, we performed a replication study in two Asian populations. METHODS: We genotyped five common variants previously reported in Europeans: rs1799884 (GCK), rs780094 (GCKR), rs560887 (G6PC2-ABCB11) and both rs1387153 and rs10830963 (MTNR1B) in the general Japanese (n = 4,813) and Sri Lankan (n = 2,319) populations. To identify novel variants, we further examined genetic associations near each locus by using GWA scan data on 776 non-diabetic Japanese samples. RESULTS: Fasting glucose association was replicated for the five single nucleotide polymorphisms (SNPs) at p < 0.05 (one-tailed test) in South Asians (Sri Lankan) as well as in East Asians (Japanese). In fine-mapping by GWA scan data, we identified in the G6PC2-ABCB11 region a novel SNP, rs3755157, with significant association in Japanese (p = 2.6 x 10(-8)) and Sri Lankan (p = 0.001) populations. The strength of association was more prominent at rs3755157 than that of the original SNP rs560887, with allelic heterogeneity detected between the SNPs. On analysing the cumulative effect of associated SNPs, we found the per-allele gradients (beta = 0.055 and 0.069 mmol/l in Japanese and Sri Lankans, respectively) to be almost equivalent to those reported in Europeans. CONCLUSIONS/INTERPRETATION: Fasting glucose association at four tested loci was proven to be replicable across ethnic groups. Despite this overall consistency, ethnic diversity in the pattern and strength of linkage disequilibrium certainly exists and can help to appreciably reduce potential causal variants after GWA studies.

2-Phenylacetylenesulfonamide (PAS) induces p53-independent apoptotic killing of B-chronic lymphocytic leukemia (CLL) cells.

We studied the actions of 2-phenylacetylenesulfonamide (PAS) on B-chronic lymphocytic leukemia (CLL) cells. PAS (5-20 microM) initiated apoptosis within 24 hours, with maximal death at 48 hours asassessed by morphology, cleavage of poly(ADP-ribose) polymerase (PARP), caspase 3 activation, and annexin V staining. PAS treatment induced Bax proapoptotic conformational change, Bax movement from the cytosol to the mitochondria, and cytochrome c release, indicating that PAS induced apoptosis via the mitochondrial pathway. PAS induced approximately 3-fold up-regulation of proapoptotic Noxa protein and mRNA levels. In addition, Noxa was found unexpectedly to be bound to Bcl-2 in PAS-treated cells. PAS treatment of CLL cells failed to up-regulate p53, suggesting that PAS induced apoptosis independently of p53. Furthermore, PAS induced apoptosis in CLL isolates with p53 gene deletion in more than 97% of cells. Normal B lymphocytes were as sensitive to PAS-induced Noxa up-regulation and apoptosis as were CLL cells. However, both T lymphocytes and bone marrow hematopoietic progenitor cells were relatively resistant to PAS. Our data suggest that PAS may represent a novel class of drug that induces apoptosis in CLL cells independently of p53 status by a mechanism involving Noxa up-regulation.

p53-mediated apoptosis of CLL cells: evidence for a transcription-independent mechanism.

The p53 protein plays a key role in securing the apoptotic response of chronic lymphocytic leukemia (CLL) cells to genotoxic agents. Transcriptional induction of proapoptotic proteins including Puma are thought to mediate p53-dependent apoptosis. In contrast, recent studies have identified a novel nontranscriptional mechanism, involving direct binding of p53 to antiapoptotic proteins including Bcl-2 at the mitochondrial surface. Here we show that the major fraction of p53 induced in CLL cells by chlorambucil, fludarabine, or nutlin 3a was stably associated with mitochondria, where it binds to Bcl-2. The Puma protein, which was constitutively expressed in a p53-independent manner, was modestly up-regulated following p53 induction. Pifithrin alpha, an inhibitor of p53-mediated transcription, blocked the up-regulation of Puma and also of p21(CIP1). Surprisingly, pifithrin alpha dramatically augmented apoptosis induction by p53-elevating agents and also accelerated the proapoptotic conformation change of the Bax protein. These data suggest that direct interaction of p53 with mitochondrial antiapoptotic proteins including Bcl-2 is the major route for apoptosis induction in CLL cells and that p53's transcriptional targets include proteins that impede this nontranscriptional pathway. Therefore, strategies that block up-regulation of p53-mediated transcription may be of value in enhancing apoptosis induction of CLL cells by p53-elevating drugs.

The efficacy of micronutrient supplementation in reducing the prevalence of anaemia and deficiencies of zinc and iron among adolescents in Sri Lanka.

OBJECTIVE: To determine the effectiveness of combined iron and zinc over the iron or zinc-only supplementation in correcting deficiency and possible interactive effects in a group of adolescent school children. SUBJECTS AND METHODS: Schoolchildren (n=821) of 12-16 years of age were randomized into four groups and supplemented with iron (50 mg/day), zinc (14 mg/day), iron+zinc or placebo capsules 5 days per week for 24 weeks. Anthropometry, and haemoglobin (Hb), serum zinc (SZn) and serum ferritin (SF) concentrations were determined before and after the intervention. RESULTS: There were no significant effects between-groups in their weight, height and Hb concentrations with the intervention when compared with the placebo group. Iron-only and combination-supplemented groups had reached mean SF concentrations of 55.1 microg/l with no difference between them (P=0.99). The zinc-only group had a mean change of 4.3 micromol//l whereas the combine-supplemented group had a mean change of 4.0 micromol/l (P=0.82). The prevalence of anaemia was found to be 70.3% in the iron group at baseline; this was reduced to 14.5% after the supplementation. In the combine-supplemented group anaemia, prevalence was reduced from 64.8 to 19.3%. CONCLUSIONS: Zinc alone or in combination with iron has not shown a significant improvement in growth in adolescence. Severe and moderate forms of anaemia were successfully treated in children who received iron supplementation. Initial high prevalence of low SZn and iron stores was significantly improved with micronutrient supplementation.

The sesquiterpene lactone parthenolide induces selective apoptosis of B-chronic lymphocytic leukemia cells in vitro.

We have studied the in vitro actions of the sesquiterpene lactone parthenolide (PTL) on cells isolated from patients with chronic lymphocytic leukemia (CLL). Dye reduction viability assays showed that the median LD(50) for PTL was 6.2 muM (n=78). Fifteen of these isolates were relatively resistant to the conventional agent chlorambucil but retained sensitivity to PTL. Brief exposures to PTL (1-3 h) were sufficient to induce caspase activation and commitment to cell death. Chronic lymphocytic leukemia cells were more sensitive towards PTL than were normal T lymphocytes or CD34(+) haematopoietic progenitor cells. The mechanism of cell killing was via PTL-induced generation of reactive oxygen species, resulting in turn in a proapoptotic Bax conformational change, release of mitochondrial cytochrome c and caspase activation. Parthenolide also decreased nuclear levels of the antiapoptotic transcription factor nuclear factor-kappa B and diminished phosphorylation of its negative regulator IkappaB. Killing of CLL cells by PTL was apparently independent of p53 induction. This is the first report showing the relative selectivity of PTL towards CLL cells. The data here warrant further investigation of this class of natural product as potential therapeutic agents for CLL.

Ligation of CD8alpha on human natural killer cells prevents activation-induced apoptosis and enhances cytolytic activity.

It has been previously shown that the subset of human natural killer (NK) cells which express CD8 in a homodimeric alpha/alpha form are more cytotoxic than their CD8- counterparts but the mechanisms behind this differential cytolytic activity remained unknown. Target cell lysis by CD8- NK cells is associated with high levels of effector cell apoptosis, which is in contrast to the significantly lower levels found in the CD8alpha+ cells after lysis of the same targets. We report that cross-linking of the CD8alpha chains on NK cells induces rapid rises in intracellular Ca2+ and increased expression of CD69 at the cell surface by initiating the influx of extracellular Ca2+ ions. We demonstrate that secretion of cytolytic enzymes initiates NK-cell apoptosis from which CD8alpha+ NK cells are protected by an influx of exogenous calcium following ligation of CD8 on the NK-cell surface. This ligation is through interaction with fellow NK cells in the cell conjugate and can occur when the target cells lack major histocompatibility complex (MHC) Class I expression. Protection from apoptosis is blocked by preincubation of the NK cells with anti-MHC Class I antibody. Thus, in contrast to the CD8- subset, CD8alpha+ NK cells are capable of sequential lysis of multiple target cells.

CCK2 gastrin receptor as a potential target for therapy in leukaemia cell lines.

The gastrin CCK2 pathway has been implicated in the development of various cancers including leukaemia. An autocrine or intracrine pathway may exist in the leukaemia cell that is involved in stimulating proliferation. We tested four leukaemia cell lines, KU812, ML-1, MOLT-4 and U937 for the existence of the CCK2 receptor and gastrin precursor protein using immunoblotting. We also assessed the effect of CCK2 antagonist PD 135 and both gastrin 17 and glycine-extended gastrin on the proliferation of the cell lines. We found immunoreactive CCK2 and gastrin precursors present in all 4 cell lines. We also observed a stimulatory effect on proliferation by gastrin and glycine-extended gastrin on 2 and 3 of the cell lines respectively and an inhibitory effect of PD 135 on all 4 cell lines. These results demonstrate that the gastrin-gastrin receptor axis is a potential target for new therapeutic strategies.

Selective apoptotic killing of malignant hemopoietic cells by antibody-targeted delivery of an amphipathic peptide.

The alpha-helical amphipathic peptide D-(KLAKLAK)2 is toxic to eukaryotic cells if internalized by a suitable targeting mechanism. We have targeted this peptide to malignant hemopoietic cells via conjugation to monoclonal antibodies, which recognize lineage-specific cell surface molecules. An anti-CD19/peptide conjugate efficiently killed 3/3 B lymphoid lines. However, an anti-CD33/peptide conjugate was cytotoxic to only one of three CD33-positive myeloid leukemia lines. The IC50 towards susceptible lines were in the low nanomolar range. Conjugates were highly selective and did not kill cells that did not express the appropriate cell surface cognate of the antibody moiety. Anti-CD19/peptide conjugates efficiently killed cells from patients with chronic lymphocytic leukemia but anti-CD33/peptide reagents were less effective against fresh acute myeloid leukemia cells. We therefore suggest that amphipathic peptides may be of value as targeted therapeutic agents for the treatment of a subset of hematologic malignancies.

Geldanamycin and herbimycin A induce apoptotic killing of B chronic lymphocytic leukemia cells and augment the cells' sensitivity to cytotoxic drugs.

We studied the actions of geldanamycin (GA) and herbimycin A (HMA), inhibitors of the chaperone proteins Hsp90 and GRP94, on B chronic lymphocytic leukemia (CLL) cells in vitro. Both drugs induced apoptosis of the majority of CLL isolates studied. Whereas exposure to 4-hour pulses of 30 to 100 nM GA killed normal B lymphocytes and CLL cells with similar dose responses, T lymphocytes from healthy donors as well as those present in the CLL isolates were relatively resistant. GA, but not HMA, showed a modest cytoprotective effect toward CD34+ hematopoietic progenitors from normal bone marrow. The ability of bone marrow progenitors to form hematopoietic colonies was unaffected by pulse exposures to GA. Both GA and HMA synergized with chlorambucil and fludarabine in killing a subset of CLL isolates. GA- and HMA-induced apoptosis was preceded by the up-regulation of the stress-responsive chaperones Hsp70 and BiP. Both ansamycins also resulted in down-regulation of Akt protein kinase, a modulator of cell survival. The relative resistance of T lymphocytes and of CD34+ bone marrow progenitors to GA coupled with its ability to induce apoptosis following brief exposures and to synergize with cytotoxic drugs warrant further investigation of ansamycins as potential therapeutic agents in CLL.

Cytotoxic drugs enhance the ex vivo sensitivity of malignant cells from a subset of acute myeloid leukaemia patients to apoptosis induction by tumour necrosis factor receptor-related apoptosis-inducing ligand.

We have studied the actions of tumour-necrosis-factor-related apoptosis-inducing ligand (TRAIL) on cells isolated from patients with acute myeloid leukaemia (AML). Apoptosis induction was initially assessed by quantitative morphological analysis. Only 2/19 isolates showed a > 10% increase in apoptotic cells following TRAIL treatment. However, incubation with TRAIL combined with fludarabine, cytosine arabinoside or daunorubicin resulted in additive or super-additive apoptosis induction in approximately half of the isolates. Molecular evidence of super-additive apoptosis induction by TRAIL and cytotoxic agents was obtained by quantification of caspase 3 activation, detected by Western blot analysis of poly (ADP ribose) polymerase cleavage. The ability of TRAIL and daunorubicin to induce super-additive apoptosis correlated with the ability of these agents to activate caspase 8 and to augment cellular levels of the truncated pro-apoptotic form of the BCL-2 family member BID. Our data suggest that co-administration of TRAIL with conventional cytotoxic drugs may be of therapeutic value in some patients with AML.

Microbiology of cerebral abscess at the neurosurgical unit of the National Hospital of Sri Lanka.

OBJECTIVES: To determine the microbial pathogens responsible for cerebral abscess, ascertain the most suitable antibiotic for treatment and to determine the predisposing causes of cerebral abscess. DESIGN: Prospective study with microbiological investigation of pus aspirated from cerebral abscesses. SETTING: Neurosurgical Unit, National Hospital of Sri Lanka, Colombo. STUDY GROUP: 41 patients with cerebral abscess. PERIOD OF STUDY: 18 months (May 1997 to December 1998) RESULTS: Of the 41 samples of pus 26 (63.1%) gave a positive microbial culture. The Gram stain of the direct smear was positive in 77% of the 26 positive cultures. The most frequently occurring species were Streptococcus milleri group (35%) followed by Staphylococcus aureus (10%). Anaerobes accounted for 23% of positive cultures. All Streptococcus milleri isolates were penicillin and cefotaxime, and all anaerobic isolates except one were susceptible to sensitive to metronidazole. 75% of Gram negative bacilli isolated were sensitive to cefotaxime. All Staphylococcus aureus isolates were methicillin resistant, but sensitive to vancomycin and chloramphenicol. Common predisposing conditions were congenital heart disease (30%), trauma (25%), middle ear disease (7%), and meningitis (7%). CONCLUSIONS: Organisms of the Streptococcus milleri group were most frequently found in cerebral abscesses. The present empirical therapeutic regime adopted in the unit which consisted of cefotaxime 1 g intravenously three times daily and metronidazole 500 mg intravenously three times daily was found to be satisfactory as a majority of the organisms isolated were sensitive to these antimicrobials. In the case of methicillin resistant Staphylococcus aureus (MRSA), it is recommended that chloramphenicol be added to the current regime in management until the antibiotic sensitivity pattern is available.