Effect of Human Milk and its Components on Streptococcus Mutans Biofilm Formation.

OBJECTIVE: This study investigated the effects of human breast milk and its components on the nutritional aspect of the caries process due to Streptococcus mutans UA159 biofilm formation. STUDY DESIGN: Human breast milk was collected from 11 mothers during 3-9 months postpartum. To test for the effect on biofilm formation, a 16-hour culture of S. mutans was treated with dilutions of human breast milk and several major components of human breast milk, lactose, lactoferrin, IgA, and bovine casein in sterile 96-well flat bottom microtiter plates for 24 hours. The biofilms were fixed, washed, stained with crystal violet, and extracted. Absorbance was measured to evaluate biofilm growth mass. RESULTS: Dilutions 1:10-1:2,560 of the human breast milk samples increased biofilm formation by 1.5-3.8 fold compared to the control. Lactoferrin decreased biofilm formation significantly in all dilutions (average milk concentration of 3 mg/ml). Lactose had no effect at average breast milk concentrations (60 mg/ml) except at its lowest concentration (15 mg/ml) where it was increased. IgA significantly decreased biofilm formation at its highest concentration of 2,400 µg/ml (average milk concentration 600 µg/ml). Casein caused significantly increased biofilm formation at all concentrations tested above the average milk content (2.3 mg/ml). CONCLUSIONS: The results of this study demonstrate an increase in S. mutans biofilm formation by human breast milk 3-9 months post partum. Among its major components, only casein significantly increased biofilm formation among the concentrations analyzed. Lactose had no effect except at 15 mg/ml. Lactoferrin and IgA significantly decreased S. mutans biofilm formation at their highest concentrations. This information expands the current knowledge regarding the nutritional influence of breastfeeding and validates the necessity to begin an oral hygiene regimen once the first tooth erupts.

A hepatitis B virus variant found in the sera of immunised children induces a conformational change in the HBsAg "a" determinant.

The emergence of variants in the outer envelope proteins of hepatitis B virus (HBV) are found among individuals vaccinated against HBV and asymptomatic carriers of the infection. For example, children in The Gambia vaccinated against hepatitis B may show serological evidence of breakthrough infections, particularly if anti-HBs antibodies induced by the vaccine are low in titre. A single-point mutation at nucleotide 421 of the S gene is associated with such breakthrough infections. In the present study, the antigenicity of variant HBV S protein expressed as HBsAg particles in a vaccinia virus expression system has been characterised using a panel of monoclonal antibodies directed against linear and conformational determinations of the S protein. A cellular ELISA procedure using expressed antigen in Vero cells revealed differences in reactivity using four of the six antibodies that had been raised against the adw subtype of HBV and recognise conformational epitopes in the a determinant. In two instances, an enhanced reactivity for the variant antigen was found, confirming that point mutations in the a determinant of the S protein between residues 139 and 147 may result in significant changes in conformation. These findings also demonstrate that there are distinct antigenic differences between the vaccine strains of HBsAg/ adw subtype and the predominant HBsAg subtype circulating in West Africa. The implications of this work are that serodiagnosis of HBV infections may be unreliable in populations where there is a possibility of variant HBV infections emerging in the face of increasing herd immunity to HBV as a result of vaccination, particularly using monoclonal antibody-based diagnostic tests. Such variants may play a role in the maintenance of HBV infections in endemic regions.

The ligase chain reaction distinguishes hepatitis B virus S-gene variants.

A novel method for the identification of hepatitis B virus (HBV) variants was developed. The ligase chain reaction (LCR) distinguished the sequences of two isolates from the Gambia showing a change at nucleotide 421 within the region coding for the surface protein a antigenic determinant. One sequence was derived from a child previously immunized with HBV vaccine, while the other, reported here for the first time, was from a chronic carrier. Nucleotide variations within the target sequence at, or up to 3 bases from the point of ligation inhibited the reaction. The LCR recognised variations in as little as 0.9 fmol DNA and will permit the rapid detection of HBV variants in molecular epidemiological studies.

Hepatitis B surface antigen variation and protective immunity.

Hepatitis B surface antigen (HBsAg) particles consist predominantly of a glycoprotein of 226 amino acids which bears the B-cell epitopes important for the induction of protective antibody responses in humans. It has been clearly shown that the region between residues 120 and 150 of the S protein represents the a determinants common to all hepatitis B virus (HBV) isolates and is exposed on the surface of the HBV particle. Anti-a antibodies protect adults against the majority of infections irrespective of the subtype of the wild-type virus. Occasional examples of infection positive for anti-HBs antibodies have been associated with the emergence of HBV variants. In particular, asymptomatic infections have been described in vaccinated children, an observation which is associated with an amino acid change in a domain critical for anti-HBs binding. Variation in amino acid sequence is also found within the preS amino terminal extensions of the S protein, although these do not correlate with subtypic variations among the S-antigenic domains. There is no direct evidence that preS determinants per se may stimulate a protective immune response in humans, although the hepatocyte attachment domain is located in the preS1 region which is conserved between HBV isolates. The inclusion of preS specificities augments anti-HBs responses in an experimental animal; however, at the present time it is unclear as to how this may best be exploited in improving hepatitis B vaccines for human use. Variability in HBV envelope proteins has implications for the design of vaccination programmes and the diagnosis of HBV infections; however, the low frequency of HBV variants emerging in the face of increasing levels of herd immunity to hepatitis B at the present time means that the extension of immunization programmes using existing vaccines remains a priority.

Monoclonal antibody TKH2 to the cancer-associated epitope sialosyl Tn shows cross-reactivity with variants of normal colorectal goblet cell mucin.

It has been suggested that synthesis of mucin-type carbohydrate chains may be arrested at the core structure Tn. This may occur following premature sialylation of Tn giving the structure STn. TKH2, a monoclonal antibody to STn, is regarded as showing highly specific reactivity with colorectal and other epithelial neoplasms. In this study we have shown that TKH2 cross-reacts with normal colorectal goblet cells that are mild PAS positive (specific for non-O-acetylated sialic acid). Approximately 9% of caucasians secrete colorectal mucin in which sialic acid is non-O-acetylated. TKH2 also cross-reacts with mild PAS negative colorectal goblet cells when sections have been pre-treated with potassium hydroxide (to remove O-acetyl groups from sialic acid). These findings make it likely that the usual non-reactivity evidenced by TKH2 for normal colorectal goblet cells is due to the presence of sialic acid that is heavily O-acetylated. The reactivity of TKH2 for colorectal cancer mucin can be readily explained by the loss of sialic acid O-acetyl substituents and not necessarily by incomplete synthesis of mucin-type carbohydrate chains.

A novel hepatitis B virus variant in the sera of immunized children.

A novel hepatitis B virus (HBV) variant was detected in the sera of two children in The Gambia, West Africa. The children had been immunized with plasma-derived vaccine and had developed antibody titres of 1448 international units x 10(-3) (mIU)/ml and 133 mIU/ml respectively against the hepatitis B surface antigen (HBsAg). Despite the protective levels of antibodies, HBV DNA was subsequently detected in both children. The complete surface (S) protein gene sequence demonstrated that this HBV isolate was closely related to the ayw4 subtype. However, five nucleotide changes were identified and two of these were unique to the Gambian isolate. One of these changes was within the region of the S gene coding for the immunodominant a determinant of the S protein. A unique nucleotide change from adenosine to guanosine at nucleotide 421 was found, resulting in an amino acid substitution at residue 141 from lysine to glutamic acid. Previous studies have shown that amino acids 141 to 146 are critical for binding to the protective anti-HBsAg antibodies. The presence of a variant HBV in these children suggests the emergence of a novel strain of HBV which can evade immune recognition. This has potential implications for HBV diagnosis and prophylaxis.

Analysis of the glycoprotein gene of Tacaribe virus and neutralization-resistant variants.

We have previously generated neutralization-resistant variants of Tacaribe virus in the presence of a monoclonal antibody (MAb) specific for the envelope glycoprotein. The envelope glycoprotein precursor (GPC) genes of two variant viruses were sequenced following polymerase chain reaction amplification of a specific region of the Tacaribe virus S RNA, and compared with the GPC gene of the parental virus. Multiple nucleotide changes in the 3' half of the GPC gene were identified in the variants, suggesting that this part of the gene codes for the envelope glycoprotein of Tacaribe virus recognized by the MAb. Both variants showed unique amino acid substitutions up to 166 residues apart, suggesting that the most likely basis for neutralization resistance was a change in an epitope in which the critical residues are juxtaposed by conformation rather than by proximity in coding.

Stability of further-attenuated measles vaccines.

Accelerated stability tests on lyophilized measles vaccines show two distinct mechanisms of virus inactivation. A rapid initial loss of infectivity occurs only on exposure to temperatures above the ambient temperature. This loss is temperature related and may be attributable to the movement of residual moisture from the virus pellet into the void space of the vial. Subsequent inactivation of virus occurs at all temperatures as a first-order reaction that follows Arrhenius kinetics. Integration of values for these two components allows precise prediction of vaccine stability at any temperature. Analysis of the results obtained for greater than 30 vaccines shows that those which are stable for one week at 37 C have a predicted life of more than one year at 8 C. This simple test is now being applied to the identification of unstable products. The rate of this reaction is closely, if conservatively, matched by a time-temperature color indicator, which may be useful for monitoring vaccine quality.

Prospects for large scale production of thermolabile products using anchorage dependent cells.

Availability of culture systems for the large scale production of anchorage dependent cells is briefly outlined. Studies on two thermolabile viruses (Measles and Influenza), showing inactivation rates of 0.1 - 0.2 log 10 per hour, clearly demonstrate the need for minimal residence time in the culture system. Logistical considerations indicate that small capacity perfusion culture systems for anchorage dependent cells would give increased recovery of these products and may readily be combined with subsequent processing steps.

Efficiency of medium utilization by human diploid fibroblasts in culture.

Growth of two human diploid cell strains were studied in relation to medium formulation, supplementation and inoculum size. MRC-5 cells were found to give greatly improved yields in Dulbecco Modified Eagle's medium, while a foreskin strain, HFS-30, showed only a slight effect. Supplementation of nutrient by the addition of fresh medium after two days incubation and the use of small inocula, gave enhanced yields and medium utilisation in both cell types.