Anti-HIV-1 activity of salivary MUC5B and MUC7 mucins from HIV patients with different CD4 counts

BACKGROUND: We have previously shown that MUC5B and MUC7 mucins from saliva of HIV negative individuals inhibit HIV-1 activity by 100% in an in vitro assay. The purpose of this subsequent study was to investigate whether MUC5B and MUC7 from saliva of HIV patients or with full blown AIDS had a similar inhibitory activity against the virus. METHODS: Salivary MUC5B and MUC7 from HIV patients with different CD4 counts ( 400) were incubated with HIV-1 prior to infection of the human T lymphoblastoid cell line (CEM SS cells). Cells were then cultured and viral replication was measured by a qualitative p24 antigen assay. The size, charge and immunoreactivity of mucins from HIV negative and positive individuals was also analysed by SDS-PAGE, Western blot and ELISA respectively. RESULTS: It was shown that irrespective of their CD4 counts both MUC5B and MUC7 from HIV patients, unlike the MUC5B and MUC7 from HIV negative individuals, did not inhibit HIV-1 activity. Size, charge and immunoreactivity differences between the mucins from HIV negative and positive individuals and among the mucins from HIV patients of different CD4 count was observed by SDS-PAGE, Western blot and ELISA. CONCLUSIONS: Purified salivary mucins from HIV positive patients do not inhibit the AIDS virus in an in vitro assay. Although the reason for the inability of mucins from infected individuals to inhibit the virus is not known, it is likely that there is an alteration of the glycosylation pattern, and therefore of charge of mucin, in HIV positive patients. The ability to inhibit the virus by aggregation by sugar chains is thus diminished

The role of crude saliva and purified salivary mucins in the inhibition of the Human Immunodeficiency Virus type 1

BACKGROUND: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. This project sought to verify statistically previous findings in our laboratory, that crude saliva from uninfected individuals together with its purified mucin components inhibited HIV-1, whilst mucins from infected saliva did not show this inhibition, in an in vitro assay. METHODS: Saliva was extracted in 4M guanidinium hydrochloride and proteolytic inhibitors at pH 6.5, followed by the isolation of MUC5B and MUC7 by Sepharose 4B gel filtration and further purification of these mucins by density-gradient ultra-centrifugation in caesium chloride. Agarose gel electrophoresis, Western blotting and amino acid compositional analysis determined the size, purity and identity of the mucins. The inhibitory activity of crude saliva and purified MUC5B and MUC7, from HIV negative (n=20) and HIV positive (n=20) donors, was tested by their incubation with subtype C HIV-1 and subsequent infection of peripheral blood mononuclear cells (PBMCs). PCR was done on tandem repeat regions of MUC5B and MUC7 DNA to investigate whether any association existed between gene polymorphism and susceptibility to infection. RESULTS: There was an inter-individual variation in the amounts of MUC5B and MUC7 in saliva. In contrast to previous studies, crude saliva and purified mucins from both HIV negative and HIV positive individuals inhibited the infection of HIV-1 in an in vitro assay. DNA analysis of the tandem repeat regions of MUC5B and MUC7 revealed no difference between groups. CONCLUSIONS: Crude saliva and its mucins, MUC5B and MUC7, from both uninfected controls and HIV positive individuals inhibited HIV-1 in an in vitro assay

The role of crude human saliva and purified salivary MUC5B and MUC7 mucins in the inhibition of Human Immunodeficiency Virus type 1 in an inhibition assay

BACKGROUND: Despite the continuous shedding of HIV infected blood into the oral cavity and the detectable presence of the AIDS virus at a high frequency, human saliva is reported to inhibit oral transmission of HIV through kissing, dental treatment, biting, and aerosolization. The purpose of this study was to purify salivary MUC5B and MUC7 mucins from crude saliva and determine their anti-HIV-1 activities. METHODS: Following Sepharose CL-4B column chromatography and caesium chloride isopycnic density-gradient ultra-centrifugation, the purity and identity of the mucins was determined by SDS-PAGE and Western blotting analysis respectively. Subsequently an HIV-1 inhibition assay was carried out to determine the anti-HIV-1 activity of the crude saliva and purified salivary mucins by incubating them with subtype D HIV-1 prior to infection of the CD4(+ )CEM SS cells. RESULTS: Western blotting analysis confirmed that the mucin in the void volume is MUC5B and the mucin in the included volume is MUC7. The HIV inhibition assay revealed that both the crude saliva and salivary MUC5B and MUC7 mucins inhibited HIV-1 activity by 100%. CONCLUSION: Although the mechanism of action is not clear the carbohydrate moieties of the salivary mucins may trap or aggregate the virus and prevent host cell entry

Review of immunological and virological aspects as contributory factors in Sudden Unexpected Death in Infancy (SUDI)

Currently in South Africa research into sudden unexpected death in infancy (SUDI) is limited. The causes of sudden infant death syndrome (SIDS) remain obscure despite full medico-legal investigations inclusive of autopsy, scene visit and ancillary studies. Viral infections play an important role as a multitude of respiratory viruses have been detected in autopsy specimens and are implicated in these deaths. The specific contribution of viruses in the events preceding SIDS still warrants deciphering. Infancy is characterised by marked vulnerability to infections due to immaturities of the immune system that may only resolve by the age of 24 months. Routine viral screening of all SUDI cases at Tygerberg Forensic Pathology Service (FPS) Mortuary in Cape Town focuses on only a portion of respiratory viruses from lung and liver tissue. This review highlights important virological and immunological aspects regarding investigations into the infectious nature of SUDI, including the lack of national standardised guidelines for appropriate specimen collection at autopsy and subsequent laboratory analysis.http://www.elsevier.com/locate/forsciint2015-12-31hb201

The inhibition of the Human Immunodeficiency Virus type 1 activity by crude and purified human pregnancy plug mucus and mucins in an inhibition assay

Background: The female reproductive tract is amongst the main routes for Human Immunodeficiency Virus (HIV) transmission. Cervical mucus however is known to protect the female reproductive tract from bacterial invasion and fluid loss and regulates and facilitates sperm transport to the upper reproductive tract. The purpose of this study was to purify and characterize pregnancy plug mucins and determine their anti-HIV-1 activity in an HIV inhibition assay. Methods: Pregnancy plug mucins were purified by caesium chloride density-gradient ultra-centrifugation and characterized by Western blotting analysis. The anti-HIV-1 activities of the crude pregnancy plug mucus and purified pregnancy plug mucins was determined by incubating them with HIV-1 prior to infection of the human T lymphoblastoid cell line (CEM SS cells). Results: The pregnancy plug mucus had MUC1, MUC2, MUC5AC and MUC5B. The HIV inhibition assay revealed that while the purified pregnancy plug mucins inhibit HIV-1 activity by approximately 97.5%, the crude pregnancy plug mucus failed to inhibit HIV-1 activity. Conclusion: Although it is not clear why the crude sample did not inhibit HIV-1 activity, it may be that the amount of mucins in the crude pregnancy plug mucus (which contains water, mucins, lipids, nucleic acids, lactoferrin, lysozyme, immunoglobulins and ions), is insufficient to cause viral inhibition or aggregation.Peer Reviewe

Delayed BCG immunization does not alter antibody responses to EPI vaccines in HIV-exposed and -unexposed South African infants.

BACKGROUND: Bacille Calmette-Guérin (BCG) is routinely given at birth in tuberculosis-endemic settings due to its protective effect against disseminated tuberculosis in infants. BCG is however contraindicated in HIV-infected infants. We investigated whether delaying BCG vaccination to 14 weeks of age affected vaccine-induced antibody responses to Haemophilus influenzae type b (Hib)-conjugate, pertussis, tetanus and Hepatitis B (HBV) vaccines, in HIV-exposed uninfected (HEU) and -unexposed uninfected (HUU) infants. METHODS: Infants were randomized to receive BCG at birth or at 14 weeks of age. Blood was taken at 14, 24, and 52 weeks of age and analyzed for Hib, pertussis, tetanus and HBV specific antibodies. RESULTS: BCG was given either at birth (106 infants, 51 HEU) or at 14 weeks of age (74 infants, 50 HEU). The timing of BCG vaccination did not influence the antibody response to any antigen studied. However, in a non-randomized comparison, HEU infants had higher Hib antibody concentrations at weeks 14 and 24 (p=0.001 and <0.001, respectively) and pertussis at week 24 (p=0.003). Conversely, HEU infants had lower antibody concentrations to HBV at 14 and 52 weeks (p=0.032 and p=0.031) with no differences in tetanus titres. CONCLUSIONS: HIV exposure, but not the timing of BCG vaccination, was associated with antibody concentrations to Hib, pertussis, HBV and tetanus primary immunization. CLINICAL TRIAL REGISTRATION: DOH-27-1106-1520

The role of occupational exposure in the development of latex hypersensitivity

Thesis (MTech (Biomedical Technology))--Cape Technikon, 2000.Professionals in a healthcare setting use latex gloves on a daily basis, primarily to prevent transmission of microbial and viral organisms to and from patients and specimens. Repeated exposure to latex proteins (through direct skin contact or mucous membrane absorption) leads to the formation of circulating latex-specific antibodies and increases the risk of sensitisation. Among all known risk groups, healthcare workers have the highest risk to develop latex hypersensitivity. Early detection of antibodies or predisposing factors (e.g. atopy or impaired skin barrier function), could assist in the identification and management of risk groups and limit possible sensitisation. An experimental group with high occupational latex exposure is compared to a control group with low or no occupational latex exposure at Tygerberg Hospital, Cape Town. A questionnaire was completed by all subjects to obtain a thorough history of past and present latex exposure and to identify other risk factors. A complete physical examination was done to evaluate clinical signs and symptoms of risk factors and latex hypersensitivity. Atopy was evaluated by means of the United Kingdom's Diagnostic Criteria for Atopy, personal and lor family history of atopy, haematogram and total serum IgE analyses. Latex-specific IgE antibodies were measured immunometrically. Skin prick tests were performed on subjects with negative in vitro results, but with predefined clinical symptoms suggestive of latex hypersensitivity. A

Clinical and laboratory investigation of latex allergy in healthcare workers

Thesis (PhD)--Stellenbosch University, 2004.ENGLISH ABSTRACT: Healthcare workers (HCWs) wear latex gloves to protect themselves and their patients against the transmission of microbial, viral and bloodborne diseases. These individuals are primarily exposed to latex via cutaneous (direct contact) and mucocutaneous (inhalation of airborne allergens on glove powder) routes. Repeated exposure leads to the formation of circulating latex-specific IgE and subsequent sensitisation with varying clinical expression. The airconditioning system of the Tygerberg Hospital (TBH) complex was investigated for the presence of aerosolised cornstarch glove powder and proteins. Dust samples were collected from 14 areas with different levels of latex glove usage. Dust samples were spectrophotometrically compared to a calibration graph of pure glove powder. The detection of starch and proteins in all the dust samples confirmed the presence of glove powder and possibly airborne latex allergens in the airconditioning ducts. As expected, the high exposure areas showed the highest concentrations of both starch and proteins. It is possible that other proteins than latex were involved, but the confirmed high level of protein contamination should be a cause for concern. Correlation between starch and protein levels was highly significant (p<0.01) in all instances. A total of 500 questionnaires were circulated for completion by HCWs from TBH. The response rate was 69.8%. After considering specific inclusion criteria, a study group of 152 individuals was compiled (28 males, 124 females). All subjects had current latex exposure and suffered from at least three pre-defined symptoms. Serum was collected from all subjects and dermal fluid from 31 subjects. Total IgE and latex specific IgE analysis were done on all serum and dermal fluid samples. Latex-specific IgE was positive (>0.35 IU/ℓ) in 23 serum and six dermal fluid samples. Skin prick tests (SPTs)for latex were done on 59 subjects with negative serum latex-specific IgE and 34 had positive results. Twelve subjects with negative latex-specific IgE and latex SPTs underwent patch tests with the European Standard Series, a piece of latex glove and glove powder in petrolatum. Three subjects had positive results to one or more of these allergens. Western blot analysis for latex was done on all positive sera and dermal fluid collected from these subjects. Western blot analysis for latex proved to be more sensitive than the capRAST, because it was able to identify specific bands in samples with negative capRAST results. All subjects showed a band for Hev b 1, which has been confirmed as a powder-bound airborne allergen. Hev b 6.01 is associated with HCWs with cutaneous symptoms and this band was recognised by 81% of the subjects. These findings confirmed that airborne and cutaneous routes are the major routes of exposure in HCWs. According to their laboratory results, subjects were divided into the following subgroups and compared statistically: Group A (serum positive, n=23), Group B (SPT positive, n=34) and Group C (negative, n=25). Group D (withdrawn, n=70) could not be used for statistical comparisons, due to incomplete results. An overall latex allergy prevalence of 38% was found. Group A differed significantly from Group B and Group C for most clinical and special investigations. Group A and B were also combined to represent all subjects with positive results (Cohort AB). The Allergy Score and Class were highly significant when Cohort AB was compared to Group C. The selection of clinical symptoms was confirmed to be relevant and work-related deterioration on any of the symptoms should bear a high index of suspicion in the evaluation of latex allergy. Numerical indices and specific symptoms showed high positive predictive values and the Allergy Score produced statistical significance in the positive subgroups when compared to the negative subgroup. Paired statistical significance was confirmed between the Allergy Score and occupational exposure (number of years, hours and pairs per week). The areas with the highest occupational latex exposure in HCWs are the face and hands. Different occupations also have different levels of exposure and two subgroups of HCWs (16 laboratory technologists and 13 theatre staff) were investigated for sebum content on different facial areas and the palms and dorsal areas of both hands. Baseline measurements were done before putting on gloves. In 21 subjects follow up measurements were done following three to four hours of occupational exposure, but before washing their hands. Baseline and follow up values were compared for all the different anatomical regions. Levels on the forehead and cheeks increased over time, while the level on the nose decreased. All hand regions decreased significantly during occupational exposure, suggesting that glove powder contributes to dryness of the skin. In conclusion, the problem posed by latex allergy will not be solved overnight and will probably remain a major occupational hazard for years to come. It is currently not possible to avoid exposure to latex, but it is imperative to institute safety measures to prevent further sensitisation in predisposed individuals and manage those already affected.AFRIKAANSE OPSOMMING: Gesondheidswerkers dra lateks handskoene om hulleself en hulle pasiënte te beskerm teen die oordrag van mikrobiale, virale en bloed-gedraagde siektes. Die lateks blootstelling vind hier hoofsaaklik plaas via kutane (direkte velkontak) en mukokutane (inaseming van lug-gedraagde allergene op hanskoen poeier) roetes. Herhaalde blootstelling veroorsaak sirkulerende lateksspesifieke IgE en sensitisasie met variërende kliniese beelde. Die lugreëlingstelsel van die Tygerberg hospitaalkompleks is ondersoek vir die teenwoordigheid van handskoenpoeier (stysel) en lateks proteïene. Stofmonsters is versamel in 14 areas wat verskillende blootstellingsvlakke verteenwoordig het. Die stofmonsters is spektrofotometries vergelyk met "n kalibrasiekurwe van suiwer hanskoenpoeier. Stysel en proteïene kon in al die stofmonsters aangetoon word en het die teenwoordigheid van handskoenpoeier en moontlike luggedraagde lateks proteïene in die lugreëlingstelsel bevestig. Soos verwag kon word, het die hoogste stysel en proteïen waardes in hoë blootstellingsareas voorgekom. Hoogs beduidende statistiese korrelasies (p<0.01) tussen die stysel en proteïenvlakke kon aangedui word in alle monsters. "n Totaal van 500 vraelyste is gesirkuleer vir voltooiing deur TBH gesondheidswerkers, waarvan 69.8% voltooide vraelyste terugontvang is. Na evaluering van insluitingskriteria, is "n studiegroep van 152 individue saamgestel (28 mans, 124 vrouens). Almal het huidige lateks blootstelling en ten minste drie het vooraf gedefinieerde simptome gerapporteer. Serum is van die hele groep versamel en dermale vog van 31 proefpersone. Totale IgE en lateks-spesifieke IgE vlakke is op alle serum en dermale vog bepaal. Positiewe resultate (>0.35 IU/ℓ) is verkry in 23 serum en ses dermaIe vog monsters. Velpriktoets vir lateks is op 59 proefpersone uitgevoer en 34 daarvan het positiewe resultate opgelewer. Twaalf proefpersone met negatiewe lateks-spesifieke IgE en velpriktoets resultate het kutane plaktoetse ondergaan met die Europese Standaard Reeks, "n stukkie lateks handskoen en handskoenpoeier in petrolatum. Drie proefpersone het positiewe resultate teen een of meer van die allergene gehad. Westerse kladanalise vir lateks is op alle positiewe serum gedoen, asook die dermale vogte van hierdie proefpersone. Westerse kladanalise vir lateks blyk baie meer sensitief te wees as die capRAST, aangesien dit spesifieke bande kon identifiseer in monsters capRAST resultate. Alle monsters het "n band getoon vir Hev b 1, "n poeier-gebinde, luggedraade allergeen. Hev b 6.01 is geassosieer met gesondheidswerkers met velsimptome en hierdie band is gevind in 81% van die monsters. Hierdie resultate bevestig dat die belangrikste blootstelling aan lateks in gesondheidswerkers deur die vel en inaseming plaasvind. Proefpersone is in die volgende drie groepe verdeel volgens laboratorium resultate en statisties vergelyk: Groep A (positiewe serum, n=23), Groep B (positiewe velpriktoetse, n=34) en Groep C (negatief, n=25). Groep D (onttrek, n=70) kon nie vir betekenisvolle statistiese vergelykings aangewend word nie, as gevolg van onvolledige resultate. 'n Finale lateks allergie prevalensie van 38% is gevind. Groep A het hoogs beduidend verskil van Groep B en C vir die meeste van die kliniese en spesiale laboratoriumondersoeke. Groep A en B is gekombineer om alle proefpersone in te sluit met positiewe resultate (Kohort AB). Die Allergie Telling en Klas van Kohort AB was hoogs beduidend in vergelyking met Groep C. Die gekose simptome is bevestig as relevant en enige werksverwante verergering van simptome moet met 'n hoë mate van agterdog bejeën word in lateks allergie. Numeriese indekse en spesifieke simptome het hoë positiewe voorspellingswaardes gelewer en die Allergie Telling was hoogs beduidend in die positiewe subgroep in vergelyking met die negatiewe subgroep. Gepaarde statistiese beduidenheid is ook gevind tussen die Allergie Telling en beroepsblootstelling (jare van blootstelling, uur en paar handskoene per week). Die meeste beroepsblootstelling aan lateks in gesondheidswerkers vind plaas op die hande en gesig. Verskillende beroepe het ook verskillende blootstellingsvlakke en two subgroepe gesondheidswerkers (16 laboratorium tegnoloë en 13 teater personeel) is ondersoek vir die sebumgehalte op veskillende areas van die gesig en hande. Basislynvlakke is gemeet voordat handskoene aangetrek is en in 21 gevalle is opvolgvlakke gemeet na drie tot vier uur beroepsblootstelling, maar voor die hande gewas is. Basislyn en opvolgvlakke is met mekaar vergelyk vir al die anatomiese areas. Die voorkop en wange het 'n toename in sebumgehalte getoon, terwyl dié van die neus afgeneem het. AI die areas op die hande toon 'n hoogs beduidende afname tydens beroepsblootstelling, wat impliseer dat hanskoenpoeier moontlik bydra tot droogheid van die vel. In samevatting, die lateks allergie probleem sal nie oornag opgelos word nie en sal waarskynlik 'n belangrike beroepsrisiko bly vir die aansienlike toekoms. Totale vermyding van lateks is tans onmoontlik en daarom is dit van uiterste belang om voorsorgmaatreëls in plek te stel om verdere sensitisasie in blootgestelde individue te verhoed en die wat reeds geaffekteer is, effektief te hanteer

Diagnostic options for investigating viral respiratory pathogens in sudden unexpected death in infancy (SUDI) cases

CITATION: De Beer, C. & La Grange, H. 2014. Diagnostic options for investigating viral respiratory pathogens in sudden unexpected death in infancy (SUDI) cases. Annals of Forensic Research and Analysis, 1(2): 1-6The original publication is available at https://www.jscimedcentral.comSudden and unexpected deaths in infants have occurred for centuries. It has generally been referred to as sudden infant death syndrome (SIDS). A new concept, called sudden unexpected death in infancy (SUDI) was introduced in 1989, which is used for all unexpected deaths in infants and babies, usually during sleep, where fatal injury can be excluded. By definition, cases that remain unexplained after thorough investigation are still classified as SIDS. Many risk factors have been associated with SUDI, e.g. poor socioeconomic conditions and prenatal care, multiple pregnancies, parental drug use and smoking, gender, low birth weight, recent infection and the sleeping environment. Ultimately, SUDI is most probably a result of a combination of predisposing factors, external stresses and underlying vulnerabilities, although the exact mechanism of death remains unknown. Viral infections are common in infants and have repeatedly been implicated in SUDI. Respiratory infections occur frequently in infancy and early childhood and inflammatory changes in the respiratory tract in SUDI cases is often found. Different diagnostic approaches for investigating respiratory viruses in SUDI cases have been reported in the literature, but in the absence of standardised SUDI investigation protocols, research from different centres cannot be compared. Viral viability is compromised in post-mortem samples and results should be interpreted with care, as the mere presence of a pathogen does not confirm that to be the cause of death. It is therefore imperative to use a combination of diagnostic approaches in parallel with epidemiological and clinical information in SUDI cases.https://www.jscimedcentral.com/Forensic/vol1issue2.phpPublisher's versio

Immune biomarkers as an adjunct diagnostic modality of infection in cases of sudden and unexpected death in infancy (SUDI) at Tygerberg Medico-legal Mortuary, Cape Town, South Africa

CITATION: De Beer, C., Ayele, B. T. & Dempers, J. 2021. Immune biomarkers as an adjunct diagnostic modality of infection in cases of sudden and unexpected death in infancy (SUDI) at Tygerberg Medico-legal Mortuary, Cape Town, South Africa. Human Pathology: Case Reports, 23:200477, doi:10.1016/j.ehpc.2021.200477.The original publication is available at https://www.clinicalkey.comPublication of this article was funded by the Stellenbosch University Open Access FundENGLISH ABSTRACT: Child mortality is a major health concern worldwide with over 4.2 million infants dying before reaching the age of one year in 2016 alone. Several international intervention initiatives have resulted in a decrease in the number of infant deaths; however, the incidence of sudden unexpected death in infancy (SUDI) and sudden infant death syndrome (SIDS) remain unacceptably high. SIDS still accounts for approximately 50–80% of SUDI cases, followed by infection. The aim of this study was to investigate a selection of immune biomarkers that are associated with an immune response in an effort to support the diagnosis of an infectious cause (“Infection”) e.g. bronchopneumonia, interstitial pneumonitis, etc., instead of SIDS in SUDI cases. C-reactive protein and 18 different cytokines were retrospectively quantified in serum collected during post-mortem investigations of SUDI cases admitted to the Tygerberg Medico-legal Mortuary in the Western Cape Province of South Africa between 2015 and 2017. Statistical comparison was done between infants with a final cause of death (COD) of Infection and SIDS to investigate any correlations between the immune markers and sociodemographic information of the groups. A p-value of < 0.0026, after Bonferroni correction for multiple comparisons, was considered as statistically significant. A total of 169 cases were included, of which 65 (38.5%) were assigned a cause of death of Infection and 104 (61.5%) SIDS by forensic pathologists. The male to female ratio of the entire group was 1:0.97 and the median age at the time of death was 9 (interquartile range [IQR] 10.9) weeks. The majority (56.8%) of deaths occurred during the colder seasons (autumn and winter) and the median post-mortem interval was 4 (IQR 3) days. No statistically significant differences were demonstrated for gender, season, sleeping position or bed-sharing between the Infection and SIDS groups. Age and interleukin-1α were identified as predictors of a COD of Infection before adjusting for the multiple comparisons problem. C-reactive protein was a statistically significant predictor of a COD of Infection even after adjusting for the effect of multiple comparisons. The COD is primarily based on histopathology of the lungs, where other causes of interstitial inflammation have been ruled out, and where there are morphological changes present suggestive of infection, but not enough evidence to assign a final COD of Infection, the cases are concluded as SIDS. These biomarkers can therefore be valuable in the investigation protocol of SUDI cases to increase the number Infection cases where the histopathology of the lungs is suggestive of, but does not support conclusive evidence of infection.https://www.clinicalkey.com/#!/content/playContent/1-s2.0-S2214330021000067?returnurl=https:%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS2214330021000067%3Fshowall%3Dtrue&referrer=Publisher's versio