Browsing by Author "Mall, Anwar Suleman"

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    The biochemical analysis of mucus and mucins in respiratory diseases with a focus on tuberculosis
    (2016) Mofokeng, Henrietta Refiloe; Mall, Anwar Suleman
    Respiratory diseases are a major cause of death in South Africa, with TB being one of the major respiratory illnesses. The respiratory tract is lined by a layer of mucus which protects the airways and lungs against injury by foreign agents. The main constituents of this layer of mucus are mucins. MUC5AC and MUC5B are the predominant respiratory tract mucins. However, little is known of the association between respiratory mucins and TB. This study aimed at describing the types and role of respiratory mucins in TB. Fifty six sputum samples, 17 tracheal aspirates and 95 bronchoalveolar lavages (BALs) were collected in 6M guanidinium hydrochloride and inhibitors. The airway mucus was divided into TB and non-TB groups. Mucins were reduced and alkylated with DTT and iodoacetamide and purified by density gradient ultracentrifugation in caesium chloride. Identification of MUC5AC, MUC5B, MUC2 and MUC7 were determined by western blotting and confirmed by immunohistochemistry. Western blot data proved the dominance of MUC5AC and MUC5B mucins in airway mucus. In comparison to the non-TB group, a higher secretion of MUC5AC than MUC5B in patients with TB was observed. MUC5AC also showed distinct behavioural characteristics in its fractionation in a caesium gradient compared to MUC5B. The presence of MUC5AC and MUC5B in different fractions suggests varying glycosylation of the mucin. Varying populations of MUC5B were observed in sputa with 3 new glycoforms shown in TB. A small group of TB patients had MUC7 in the sputa (and not in the lavage) and there were varying amounts of MUC2 in some TB samples and non-TB mucus. At tissue level, MUC5B was found to be the main secreted gel-forming mucin. MUC5B and MUC7 were found to play a role in the protection again infection by Mycobacterium tuberculosis in tuberculous granulomas. Using proteomics it was demonstrated that respiratory mucus protein expression differs in, tracheal aspirates, BALs and sputa. Although inter-individual variations were observed in all samples, similar proteins were expressed in relation to the functioning of the lung. O-glycan analysis showed that the majority of the O-glycans detected were sialylated and that core 3 and 4 O-glycan structures diminished in the presence of HIV.
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    The inhibition of HIV-1 activity by crude mucus and purified mucin (mucous glycoproteins) from saliva, breast milk and the cervical tract of normal subjects, HIV positive individuals and patients with HIV-AIDS
    (2007) Habte, Habtom Haileselassie; Mall, Anwar Suleman
    Human saliva, breast milk and cervical secretions contain several non-immunological components including mucins (mucous glycoproteins), which protect the gastrointesinal and female reproductive tracts and breast fed infants from bacterial, viral and fungal infections. In addition to their well known function in lubrication, tissue coating and digestion, mucus and mcins have been used as pathological markers in diseases such as asthma, chronic bronchitis, cystic fibrosis, and carcinomas of the breast, lung and colon. Crude saliva is a also known to inhibit the activity of human immunodeficiency virus (HIV), the causative agent of acquired immunodeficiency syndrome (AIDS). According to the joint United Nations Programme on HIV/AIDS (UNAIDS) worldwide an estimated 38.6 million people were living with HIV in 2005 with 401 million newly infected and 2.8 million deaths. It has been reported that an estimated 24.5 million of the HIV infected people of whom 60% females live in sub-Saharan Africa with the Southern African region having the highest prevalence in Africa. Furthermore the incidence of opportunistic diseases such as TB is also reported to increase with HIV prevalence. Thus far, despite the discovery of highly active antiretroviral therapies which contain both protease and reverse transcriptase inhibitors, HIV remains as a global threat especially to the third world countries. Therefore there is a need for the development of safe compounds to reduce viral loads in infected people and to prevent the transmission of the virus from one individual to another. The search for a suitable vaccine is ongoing.
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    An investigation of the development of a biochemical and clinical marker for gastric disease
    (2014) Chivaura, Rufaro; Mall, Anwar Suleman
    Includes abstract. Includes bibliographical references.
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    The isolation, purification, tissue localization and identification of a glycoprotein found in the crude mucus gel of patients with carcinoma of the stomach
    (2008) Nthato, Chirwa; Mall, Anwar Suleman
    The thin layer of crude mucus lining the human gastric mucosa protects the delicate gastric epithelium from the high shear forces associated with digestion. Gastric mucus is composed largely of water (>90%) and a complex mixture of organic components such as enzymes, various serum and cellular macromolecules, sloughed cells, bactericidal proteins, plasma proteins, inorganic ions and very importantly the mucins, that impart to it its gel-forming properties. Mucins are large heterogenous polymers that are difficult to characterize by traditional biochemical methods. However mucins from different regions of the body do share common features such as a low protein content of approximately 20% by weight and a carbohydrate content of 70 to 80% by weight. Mucins are characterized by a variable number of tandem repeat regions rich in serine, threonine and proline with the serine and threonine being potential sites for O-glycosylation. In contrast to the glycosylated region is the 'naked' region rich in cysteine and susceptible to proteolysis. The cysteine residues enable mucin monomers to form polymers by disulphide bridges. Alterations of mucin expression take place in gastric carcinomas. Our laboratory previously reported the presence of a 40-50 kDa glycoprotein in the mucus of patients with gastric cancer that associated with albumin. The primary aims of our study were to develop an antibody to this 40-50 kDa glycoprotein, and to determine the location of its expression in gastric tissue by the immunohistochemical method, ranging from normal, to premalignant to cancer. The final aim was to identify this unknown glycoprotein by proteomic analysis. The reactivity of the antibody to the mucin and its 40-50 kDa component was determined by Western Blotting.
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    The role of crude saliva and purified salivary mucins in the inhibition of the Human Immunodeficiency Virus type 1
    (2011) Peacocke, Julia Helen; Mall, Anwar Suleman
    Sub- Saharan Africa is the world's worst HIV-AIDS affected region. More interventions to manage this pandemic are urgently required. Transmission of the virus through an exchange of saliva is rarely known to occur. The role of saliva and its mucus in this inhibition requires clarification. This project sought to verify the previous findings in our laboratory (Habte et al. , 2006; Peacocked et al. (manuscript under revision)), that crude saliva from uninfected individuals together with its purified mucin components inhibited HIV-1 in an in vitro assay. Mucins from infected saliva did not show inhibition in this assay. Mucus was extracted in 6M guanidinium hydrochloride and a cocktail of protease inhibitors, pH 6.5. Sepharose CL-4B gel filtration separated MUC5B and MUC7 in saliva. Mucins were purified by density gradient ultra-centrifugation in caesium chloride. Western blotting and amino acid analysis determined the size, charge and identity of the mucins. The inhibitory activity of crude saliva, salivary mucin components MUC5B and MUC7 from HIV-negative and HIV-positive donors, was tested by their incubation in an in vitro HIV-neutralisation assay using a pseudovirus of Subtypes A and C, and infection of susceptible epithelial tumour cells (genetically modified TZM- bl cells). Glycosylation analysis by HPLC was also performed on each group. The presence of MUC5B and MUC7 in saliva was confirmed and it was shown that there was inter-individual variation in their amounts and electrophoretic behaviour between and within groups. Crude HIV-negative and HIV-positive saliva and its purified mucins MUC5B and MUC7 significantly inhibited the infection of HIV-1 in an in vitro pseudoviral assay in a dose-response manner. HPLC revealed two glycoforms of salivary MUC5B.
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    The role of human breast milk mucus and mucins in HIV-AIDS
    (2012) Mthembu, Yolanda; Mall, Anwar Suleman
    Milk molecules such as mucins, antibodies, bactericidal enzymes like lysozymes and fatty acids that lyse bacteria, viral particles and bacterial peptides, offer anti-microbial activity in milk. Despite human breast milk being rich in anti-microbial substances, such as mucin, that protect against pathogens and viruses, it remains a significant route of HIV transmission from mother to child. ... The objectives of the study were to isolate, purify, identify and investigate the anti-HIV-1 activity of crude breast milk particularly the human milk fat globule material (MFGM) and its purified mucin components, in HIV positive patients (n = 20) compared with those who are not infected (n = 20). This study also tested the effect that heat (80°C, 10 min) might have on breast milk which might release the milk mucins and consequently have an inhibitory effect on HIV-1.
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    Separation and purification of mucins and tenascin-C in breast milk of patients and the investigation of the role of mucins and tenascin-C in the inhibition of HIV-1
    (2017) Kehoe, Kathleen; Mall, Anwar Suleman
    An estimated 36.7 million people were living with HIV in 2015, with 2.1 million newly infected people with HIV in 2015 worldwide. The highest prevalence of HIV in 2015 was in the Eastern and Southern African regions. This highlights the importance for research in this field to further prevent the number of new HIV cases. Mother-to-child transmission of HIV is due to either cell-associated or cell-free virus present in the breast milk of an HIV positive mother. Most often, HIV positive mothers choose to breastfeed their infants due to the nutritional and immunological benefits outweighing that of the risk of HIV transmission. Importantly, approximately 85% of infants do not acquire HIV through daily exposure to breast milk from their HIV positive mothers who are not on ART suggesting that human breast milk has antiviral properties. Previously in our laboratory, MUC1 and MUC4 has been implicated in the inhibition of HIV-1 in an in vitro assay. Furthermore, crude breast milk was tested in this assay showing strong HIV-1 neutralisation. Pasteurisation (80°C for 10 minutes) of both HIV positive and negative breast milk indicated good neutralisation of HIV-1 in our laboratory. Another breast milk protein, tenascin-C (TNC), was recently shown to strongly neutralise HIV-1 in a study performed by another group. Therefore, with this knowledge, this study was employed to firstly compare the antiviral properties of MUC1, MUC4 and TNC. Furthermore, the HIV-1 neutralisation ability of crude breast milk was sought to be investigated along with the investigation of two different pasteurisation methods including 80°C for 10 minutes and 62.5°C for 30 minutes (Holder pasteurisation). Human breast milk was separated into milk fat and skim milk using caesium chloride density gradient ultracentrifugation. MUC1 was purified from the milk fat using gel extraction from a 4-20% sodium dodecyl sulphate polyacrylamide gel. The skim milk was chromatographed on a Sepharose 2B-CL column from which the void volume was collected to purify TNC using gel extraction from a 4-20% sodium dodecyl sulphate polyacrylamide gel. During this purification, a band consisting of MUC1 which adhered to TNC was used to co-purify the MUC1/TNC glycoprotein using gel extraction. MUC1 and TNC were individually purified using gel extraction. MUC4 was not successfully purified and from ELISA data it was concluded that the concentration of MUC4 was below the detectable limit of the ELISA kit. The average concentration of MUC1 was determined to be 307.85 ng/ml, while the concentration of TNC could not be determined due to the majority of absorbance values (450 nm) lying above the upper limit of the curve. The HIV neutralisation of each of the samples was tested in an in vitro HIV-1 assay. This assay utilises a luciferase reporter gene in modified TZM-bl/JC cells using Du422.1 virus derived from clad C of HIV-1. These assays are being performed to assess the antiviral properties of crude and heat treated breast milk and purified MUC1 and TNC separately as well as co-eluted and co-purified MUC1/TNC. The two pasteurisation methods increased the HIV-1 neutralisation when compared to crude breast milk. The HIV-1 neutralisation of these groups were compared with a Kruskal Wallis test and a statistically significant difference was detected among the crude and 62.5°C heat treated breast milk cohorts (Mann-Whitney U, p-value = 0.0021). Furthermore, a statistically significant difference in the HIV-1 neutralization was detected among the 80°C and 62.5°C heat treated breast milk cohorts (Mann-Whitney, p-value = 0.0033). From the data, and the range of IC₅₀ values (50% inhibitory concentration), the HIV-1 potency was deemed the strongest in the 62.5°C heat treated breast milk. This pasteurisation method could potentially be promoted in lower resource settings to decrease mother-to-child transmission of HIV-1. Purified MUC1 and TNC, as well as co-eluted and co-purified MUC1/TNC, was tested in the same neutralization assay in order to compare the HIV-1 potency of these glycoproteins. The difference in HIV-1 neutralisation was not statistically significant among all three groups (Kruskal Wallis, p-value = 0.13). From the range of IC₅₀ values, it was suggested that TNC has a stronger HIV-1 potency when compared to MUC1. Overall, the co-eluted and co-purified MUC1/TNC showed a lower HIV-1 potency when compared to the single, purified glycoprotein. MUC1 and TNC could be purified and cloned to aid in the protection against contracting HIV-1, especially in mother-to-child transmission of HIV-1. Histochemistry and immunohistochemistry was performed on breast tissue sections to investigate the morphology of the cells and the presence of MUC1, MUC4 and TNC respectively. The lactating breast tissue was confirmed to have wide, dilated lumina and vacuolated cytoplasm with neutral and sialomucins. The presence of MUC1, MUC4 (β subunit) and TNC were confirmed in the lactating breast tissue using immunohistochemistry.
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    The effect of reduction, trypsin digestion and de-glycosylation of salivary mucins in the inhibition of human immunodeficiency virus type 1
    (2019) Tsetse, Ellis CT; Mall, Anwar Suleman
    In 2017, 36.7 million people worldwide were living with Human Immunodeficiency Virus (HIV) and of that total, 1.8 million people were new infections. Sub-Saharan Africa was recognized as the most afflicted regions worldwide accounting for 26 million people, 68%, living with HIV. The difficulty in fighting this epidemic has raised the urgent need for research exploring ways in which HIV transmission can be curbed worldwide. Our laboratory previously showed that crude saliva and purified salivary mucins (MUC5B and MUC7) inhibit HIV-1 infection in vitro. However, it is not known whether the specific arrangement of mucin carbohydrate residues is important for mucin interactions with HIV-1, or if the negative charge afforded by sialic acid and sulfated sugars allows binding to viral receptors. While giving some important insight into the mechanism of HIV inhibition, we hope that this study will determine the minimum peptide chain length and structure of a gel forming mucin that retains the anti-HIV activity. In addition, we aim to determine if the reduced salivary subunits and trypsin digested fragments retained this inhibitory activity against HIV-1. Saliva was collected and stirred overnight in 6M guanidine hydrochloride with 10mM Na2HPO4, 10mM EDTA, 1mM PMSF and 5mM NEM. Salivary mucins (MUC5B and MUC7) were purified using caesium chloride ultracentrifugation and separated on a Sepharose CL-4B column. Thereafter, mucin rich fractions were either reduced with 10mM dithiothreitol (DTT) or proteolytically digested with 0.25% trypsin. The resultant fractions were dialysed and freeze dried. Slot blots were used to determine the identity of the void volume (Vo) fractions and the included volume (Vi) fractions which were identified as MUC5B and MUC7 respectively. The Vo and Vi fractions were subjected to 4- 20% sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE) to determine the size and mucin concentration. In addition, mucin oligosaccharides were enzymatically removed using the de-glycosylation kit (EDEGLY) purchased from Sigma Aldrich (UK). Following this, all mucin lyophilized aliquots were tested for cell cytotoxicity using an MTT assay. This was then followed by a neutralisation assay which uses HIV-1 env pseudo virus (DU422.1 and YU2 subtype C and subtype B respectively) and a luciferase reporter gene involving modified TZM-bl/JC cells was used to test the inhibitory activity of the test samples. Comparison of the anti-HIV activity of crude saliva, MUC5B and MUC7 against the DU422 virus showed that both crude and purified saliva indeed inhibits the infection of the DU422.1 pseudo-virus strain to TZM-bl/JC cells (Kruskal-Wallis, p=0.00025). MUC5B was more potent in inhibiting the DU422 virus as compared to crude saliva and MUC7 (MannWhitney U, p=0.0227 and p=0.0195 respectively). Furthermore, no difference was observed in inhibiting the DU422 virus by MUC7 and crude saliva (Mann-Whitney U, p=0.128). While the three cohort of samples did inhibit the YU2 pseudo virus (KruskalWallis, p=0.0078), MUC7 showed a higher inhibition compared to MUC5B and crude saliva (Mann-Whitney U, p=0.0341 and p=0.176 respectively). A significant difference in the inhibition of the YU2 virus was detected between MUC7 and crude saliva (MannWhitney U, p=0.0031). In addition, reduced and digested salivary fragments inhibited both viruses suggesting the possibility that even when the gel forming properties of mucins are compromised, mucins still retain their inhibitory activity. Interestingly, the removal of oligosaccharides showed MUC5B as the most potent mucin in the inhibition of both DU422 and the YU2 pseudo virus (Kruskal-Wallis, p=0.0312). Deglycosylated MUC7 displayed minimal inhibition against the YU2 and DU422 virus suggesting that oligosaccharides are important for maximal inhibition. Furthermore, this highlights that the mechanism through which mucins inhibit viruses involve glycans. In conclusion, the results of this study suggest that MUC5B can be harnessed and used as a core component of a microbicide which can be used to prevent HIV transmission. Its extensive glycosylation compared to MUC7 makes it a better candidate for this anti-HIV1 inhibitory activity.