Design and methodology of a study on colorectal cancer in Johannesburg, South Africa.

BACKGROUND AND AIM: Cancer is one of the foremost causes of morbidity and mortality worldwide. Globally, colorectal cancer (CRC) is the third most diagnosed and fourth most important cause of cancer death. A total of 70% of all CRC-related deaths occur in low- and middle-income countries. In Sub-Saharan Africa (SSA), estimating the burden of CRC is difficult. Only 27 of 43 SSA countries have formalized cancer registration systems; data quality is variable and national coverage rare. METHODS: This is a multidisciplinary, longitudinal cohort study started in January 2016. Patients >18 years with histologically confirmed primary adenocarcinoma of the colon and rectum, diagnosed within the previous 12 months, are eligible. Participants were assessed and were followed up for 3 years. Baseline information, including demographics, socioeconomic status, family history, medical and surgical non-cancer-related history, dietary history, colonoscopic findings, staging at presentation, treatment, and disease recurrence, is collected, as well as blood tests and histology results. Outcomes include disease recurrence (local and metastatic) and survival. RESULTS AND CONCLUSION: This study aims to describe the clinical presentation, management, and outcomes of adults with CRC in a multiethnic, urban South African population. It will be the first prospective study to describe clinical presentation, demographics, risk factors, treatment, and outcomes according to population group, from both private and state health-care facilities in Johannesburg, South Africa. The results of this study will be relevant not only to South Africa but also to other SSA countries undergoing similar rates of rapid urbanization and epidemiological transition.

Mseleni joint disease: a potential model of epigenetic chondrodysplasia.

OBJECTIVE: In this paper past research on the natural history of Mseleni joint disease, a crippling endemic osteoarthritis, its socio-economic impacts, the demographics, diet, geology and the genetic background of affected people are reviewed. In addition, some new research ideas are suggested to continue the search for etiological avenues for this disease such as stable isotope analysis and epigenetic mechanisms. RESULTS: Mseleni joint disease is a chondrodysplasia first described in 1970. It is geographically confined to a remote area in the Maputaland region in northern Kwazulu Natal, South Africa. This disease affects most joints but primarily those of the hip; it is a progressive condition beginning with pain and stiffness until the patient's ability to walk becomes compromised. Mseleni joint disease is characterized by two distinct abnormalities, protrusio acetabuli that mainly affects females and increases in frequency with age, and hip dysplasia that is more frequent with age. Much research has been conducted on the people with the disease and their surrounding environment. CONCLUSION: Despite intensive investigations into the etiology of Mseleni joint disease, it remains unknown. As a result the examination of epigenetic mechanisms and stable isotope analysis of teeth are suggested as a means of providing information on the etiology of the disease. These methods can also be applied to other chondroplasias of unknown etiology.

Brief communication: Minimally invasive bone sampling method for DNA analysis.

Obtaining a bone sample for DNA analysis has traditionally been a destructive practice, which has resulted in reluctance on behalf of curators for skeletal collections to allow invasive testing. A novel minimally invasive bone sampling method for DNA analysis is presented here. This method uses a conventional hand drill wherein the bone sample is extracted from the intercondylar fossa of the femur; it does not interfere with any known anthropometric landmarks and only leaves a small hole on the surface of the bone. The temperature of the drill is documented and it was established due to the minor increase in temperature, that this should not affect the molecular integrity of the sample. This method is easily replicated and is suitable for both human and other animal skeletal material and can be applied to rare specimens with little risk.

Novel methods of molecular sex identification from skeletal tissue using the amelogenin gene.

Sex identification from skeletal material is of vital importance in order to reconstruct the demographic variables of an individual in forensic genetics and ancient DNA (aDNA) analysis. When the use of conventional methods of sex identification are impossible, molecular analysis of the X and Y chromosomes provides an expedient solution. Two novel systems of molecular sex identification suitable for skeletal material using the amelogenin gene are described, beginning in intron 2-3, spanning exon 3 and ending in intron 3-4. This area was optimal for sexing, as it includes 14 sex-specific polymorphic regions in addition to an indel (insertion or deletion of nucleotides). Once optimised and working with 100% efficiency on the controls, these procedures were applied to a collection of miscellaneous archaeological skeletons (ex situ) sourced from the Raymond Dart Collection of Human Skeletons (Dart Collection). This collection was used to optimise these techniques for skeletal remains derived from an archaeological context. These methods produced 46.66% sex results for the ex situ sample, which is within the normal range for aDNA studies. These new techniques are optimal for sex identification, with both the inherent control of isolating many sex-specific features and combined with the use of sensitive micro-fluidic electrophoresis.

Actin binding protein expression is altered in uterine luminal epithelium by clomiphene citrate, a synthetic estrogen receptor modulator.

Clomiphene citrate (CC), a synthetic oestrogen, is often prescribed as a superovulator in treating infertility. Although CC works efficiently, pregnancy rates following CC treatment are approximately 10 times lower than "natural" rates. This study investigates how a dose of 1.25 mg CC given to ovariectomized rats before the implantation priming hormones (a single dose of progesterone for 3 days and a dose of estradiol-17beta on d3, P-P-PE), alters the expression and distribution of alpha-actinin, gelsolin and vinculin. Actin binding proteins show a specific distribution within the uterine epithelium during implantation, linking the actin cytoskeleton to integrin expression on the uterine surface and in this way aiding "adhesiveness" for blastocyst apposition to the uterine epithelium. In this study, immunocytochemistry on frozen uterine sections using mouse monoclonal antibodies against alpha-actinin, gelsolin and vinculin and peroxidase-conjugated secondary antibodies, show that CC, administered before the P-P-PE regimen, down-regulates the expression of vinculin, does not alter the expression of gelsolin and up-regulates alpha-actinin on the uterine apical surface, when compared to P-P-PE treated animals. All three proteins are down-regulated on the apical surface of the luminal epithelium and glands in all groups when compared to pregnant controls. Vinculin was only localized in the basolateral compartment of the uterine epithelial cells in the CC treated groups. By down-regulating these proteins on the uterine surface and up-regulating vinculin on the basolateral membrane of the epithelium, CC may impede adhesion and invasion of blastocysts at implantation. These results may aid the exogenous manipulation of uterine tissue to control fertility and improve assisted reproductive out-comes.

A high resolution SEM study of the effects of RU486, used as a postcoital contraceptive, on the rat uterus during early pregnancy.

During the window of receptivity, a narrow range of time under the control of the ovarian hormones progesterone and oestrogen, when a blastocyst can attach to the uterine surface, the plasma membrane of the uterine epithelial cells undergoes a remarkable change in structure, known as 'the plasma membrane transformation' of early pregnancy. RU486, the controversial abortion drug (Mifegyne), acts as a progesterone receptor antagonist, resulting in transcriptionally inactive progesterone receptors. In view of this, a change in the well-documented sequences of the plasma membrane transformation is postulated. This study therefore aims to investigate the effects of RU486 on this sequence of events in the implantation and non-implantation sites of the rat uterus. In both RU486 treated and control animals, on days 4.5, 5.5 and 6.5 of pregnancy, scanning electron microscopy revealed a distinct pattern of folding of the uterine surface in non-implantation sites. In contrast, folding was not observed within the implantation sites. These results indicate that surface alterations are probably not under the control of progesterone signalling. The lack of folding at the implantation sites possibly ensures maximum close contact between the blastocyst and the maternal tissue thus promoting implantation. During early pregnancy, specifically on day 5.5, the microvilli of the uterine epithelial cells in the treated animals were more dense than those in the untreated animals. Such microvilli are characteristic of the uterine epithelial cells of a uterus under-stimulated by hormones. Flattening of the apical cell borders usually seen at the time of blastocyst attachment and implantation was not observed following RU486 treatment. Large apical protrusions were observed in the RU486 treated animals only, possibly linked in some way to apoptosis. The antiprogestin properties of RU486 may further elucidate the progesterone effects associated with early pregnancy.

The relationship between alkaline phosphatase activity and intracellular lipid accumulation in murine 3T3-L1 cells and human preadipocytes.

Alkaline phosphatase (ALP) is expressed in 3T3-L1 preadipocytes, and its activity increases during adipogenesis. The purpose of this study was to determine whether ALP activity could be used as a measure of intracellular lipid accumulation in human preadipocytes and 3T3-L1 cells and which of the factors that induce adipogenesis are responsible for stimulating ALP activity. Adipogenesis was initiated in 3T3-L1 cells by incubation with differentiation medium containing insulin, dexamethasone, and 3-isobutyl-1-methylxanthine. The effect of leaving out each of the differentiation medium components was studied. Adipogenesis was also assessed in human preadipocytes and 3T3-L1 cells in the presence of the ALP inhibitor histidine. ALP activity was measured using an automated colorimetric assay and intracellular lipid accumulation was measured using the lipid-specific dye oil red O. Removal of insulin or dexamethasone from the differentiation medium had little effect on either ALP activity or lipid accumulation in 3T3-L1 cells, while removal of IBMX blocked both. Histidine inhibited ALP activity and adipogenesis in human preadipocytes and 3T3-L1 cells. Pearson univariate correlation analysis demonstrated strong correlations between ALP activity and lipid accumulation in human preadipocytes (r=0.78, n=69) and in 3T3-L1 cells (r=0.92, n=27). These data suggest that ALP and fat storage are tightly linked during preadipocyte maturation and that the measurement of ALP activity may be a novel technique for the quantification of intracellular lipid accumulation that is more sensitive and rapid than currently used methods.

The effect of alkaline phosphatase inhibitors on intracellular lipid accumulation in preadipocytes isolated from human mammary tissue.

BACKGROUND: A previous study has demonstrated that alkaline phosphatase (AP) may play a role in the control of intracellular lipid accumulation in the rodent preadipocyte cell line, 3T3-L1. The present study investigated whether AP may have a similar function in preadipocytes isolated from human mammary gland tissue. METHODS: Preadipocyte maturation was induced in the presence or absence of the tissue non-specific AP inhibitors levamisole and histidine, and the tissue-specific AP inhibitor PheGlyGly. Cellular AP activity and adipogenesis were both assessed at 0 and 12 days post-induction of differentiation. RESULTS: After differentiation, AP activity increased 5.1 +/- 1.3-fold in the absence and 8.9 +/- 2.8-fold (P < 0.05) in the presence of levamisole. However, adipogenesis increased 1.95 +/- 0.11-fold in the absence but only 1.36 +/- 0.06-fold (P < 0.001) in the presence of levamisole. There was a 4.2 +/- 2.2-fold increase in AP activity in the absence and a 0.51 +/- 0.46-fold (P < 0.05) decrease in the presence of histidine. Adipogenesis increased 2.09 +/- 0.35-fold in the absence of histidine but only 1.22 +/- 0.30-fold (P < 0.05) in the presence of histidine. PheGlyGly had no effects. Fluorescent microscopy showed AP activity was localized to the triglyceride-containing droplets of the cell. CONCLUSION: This is the first study to show that tissue non-specific AP inhibitors can block adipogenesis in human preadipocytes.

Alkaline phosphatase is involved in the control of adipogenesis in the murine preadipocyte cell line, 3T3-L1.

OBJECTIVE: As alkaline phosphatase may play a role in cell differentiation, our aim was to study the possible role of this enzyme in the differentiation of preadipocytes (3T3-L1 cells) into adipocytes. RESEARCH METHODS AND PROCEDURES: 3T3-L1 cells were grown in medium containing insulin, dexamethasone and IBMX to induce adipogenesis. Adipogenesis was measured using the triglyceride-specific dye, oil red O at 0, 3, 7 and 11 days after initiation of adipogenesis in the presence or absence of the alkaline phosphatase inhibitors, levamisole, histidine and Phe-Gly-Gly. Intracellular localisation of the enzyme was detected using ELF-phosphatase, a fluorescent substrate and alkaline phosphatase gene expression was assessed using RT-PCR. RESULTS: Alkaline phosphatase activity was detected in untransformed cells (1.91+/-0.62 mU/mg protein) and activity increased 11.5+/-1.4-fold after 11 days treatment with transformation medium and 5.3+/-0.3-fold in transformation medium containing levamisole (p<0.05). Triglyceride content of cells increased 3.1+/-0.2-fold after 11 days treatment with transformation medium and 2.1+/-0.3-fold in the presence of levamisole (p<0.005). Histidine inhibited adipogenesis and alkaline phosphatase to a greater extent than did levamisole, but Phe-Gly-Gly had no effect on these variables. Alkaline phosphatase was localised around the lipid droplets of the cells. Gene expression of alkaline phosphatase increased during adipogenesis. DISCUSSION: This study demonstrates that tissue-nonspecific alkaline phosphatase is present in 3T3-L1 cells and that it may play a role in the control of adipogenesis.

Retinoic acid increases the length and volume density of ducts in the rat embryonic pancreas.

In this study, the role of all-trans retinoic acid (RA) on the proliferation of rat embryonic pancreas ducts and on the proportion of insulin cells was investigated. All-trans RA (10-6 m) was added to Ham's F12.ITS serum-free medium in which 12.5 day rat dorsal pancreatic buds were cultured on Matrigel. Control explants were cultured on Matrigel in Ham's F12.ITS alone or in Ham's F12.ITS containing ethanol (the diluent for RA). After a 7 day culture period, explants were incubated with bromodeoxyuridine (BrdU) for assessment of cell proliferation. Explants were processed for both morphometry and immunocytochemistry. The length density and volume density of the pancreatic ducts were assessed using an image analysis system. Cells positive for insulin, BrdU and glucagon were localized on adjacent serial sections. RA treatment caused a statistically significant increase in the volume density (P < 0.007) and length density (P < 0.008) of the ducts, as well as a 1.2-fold increase (P < 0.0001) in the proportion of insulin to glucagon cells, compared to both control groups. Few insulin cells were BrdU positive, indicating that cells had a low proliferation rate. The increased proportion of insulin cells may relate to the increased volume density and length density of the ducts in RA-treated explants. It is suggested that RA stimulated the production of additional progenitor cells and not proliferation of existing insulin cells.

Exogenous activin increases the proportion of insulin cells in the developing chick pancreas in culture.

As activin is believed to be a key signalling factor during early pancreatic development, its influence on the proliferation and/or determination of insulin cells in the developing chick dorsal pancreatic bud was investigated. Dorsal pancreatic buds of 5-day-old chick embryos were explanted on to Matrigel and cultured in serum-free medium (Ham's F12.ITS), to which 1 or 10ng/ml activin was added. After 7 days in culture, the explants were processed for immunocytochemistry and the insulin-positive cells were scored and expressed as a proportion of the sum of insulin and glucagon cells. When compared to the control cultures (Hams F12.ITS alone), activin treatment resulted in respective increases in the proportion of insulin cells of 1.6 and 1.9 fold. It is suggested that activin treatment favours differentiation of the insulin cell pathway relative to glucagon cells.

Absolute numbers versus proportions in the assessment of differentiation of beta-cells in the embryonic avian pancreas.

Environmental factors may influence the proliferation and differentiation of embryonic pancreatic endocrine cells, creating a need for the quantification of such effects. The explanted dorsal pancreatic bud (DPB) of the 5-day chick embryo is a useful in vitro model. Since all explants cannot be assumed to have the same number of endocrine cells at the start of culture, the proportion of beta-cells with respect to alpha-cells may be a more meaningful measure than absolute numbers. This study aimed to establish baseline values for the proportion of beta-cells in both intact and mesoderm-depleted DPBs before culture. Buds were excised from 12 chick embryos and the surrounding mesoderm was removed from 6 buds following collagenase treatment. All the buds were freeze-dried, fixed in parabenzoquinone vapour, embedded in resin and sectioned at 1 micro m. alpha- and beta-cells were detected by an indirect immunoenzyme method. alpha-cells outnumbered beta-cells in 9 of the 12 buds. The proportion of beta-cells in the intact buds varied from 16% to 64% (mean 39.5%) and in the mesoderm-depleted buds from 17% to 66% (mean 39%). There was no significant difference between the absolute numbers or the proportions of cells in either case. The proportions of beta-cells in the 5-day DPBs were higher than those in buds cultured in previous studies for 7 days under various conditions. This result may reflect the role of apoptosis in response to the culture conditions.