Lectin binding affinities of human milk fat globule (HMFG) membrane antigens.

Six biotinylated lectins with differing specificities and two monoclonal antibodies (III D 5 and III H 2) were used to characterize the sugar-residues in human milk fat globule (HMFG) membrane antigens. Immunoblotting analysis revealed that most of the antigens contain several sugars. However, the molecules exclusively reacting with anti-HMFG III D 5, a monoclonal antibody previously shown to detect antigen(s) positively correlating with the expression of estrogen receptors in mammary and gynaecological carcinomas, could only be stained with peanut agglutinin and Ricinus communis-lectins. One of these antigens, a 42-57 kDa molecule, was shown to have a complexed quaternary structure with galactose determining the antigenic specificity. It is suggested that the production of this glycoprotein in estrogen sensitive tissues results from activation of galactosyl-transferase-enzyme at the same time as the expression of estrogen receptors.

One- and two-step non-competitive avidin-biotin immunoassays for monomeric and heterodimeric antigen.

In this study of one-step and two different two-step non-competitive avidin-biotin assays (NABAs) were developed for the measurement of a monomeric antigen (lactoferrin, LF) using polyclonal antibodies and the detection of a heterodimeric antigen (lutropin. LH) using monoclonal antibodies. The assays were based on the use of performed complexes of biotinylated antibody and avidin-peroxidase conjugate. The detection limits and intra-assay CVs of the one- and two-step NABAs were 0.1-0.5 mg/ml and 2.6-5.1% for LF, and 0.1-0.2 IU/l and 2.3-3.7% for LH, respectively. The working range was 1-100 ng/ml for the LF assay and 1-100 IU/l for the LH assay. A linear relationship with high correlation coefficients (0.979-0.992 for LF-NABAs: 0.949-0.990 for LH-NABAs) and good agreement was observed between the one- and two-step assays and the corresponding three-step NABAs used as reference methods. However, under stringent conditions the one-step assay for heterodimeric antigen was found to be sensitive to interference. The results indicate that it is possible to perform the multistep NABAs using convenient one- and two-step protocols. The one- and two-step assays also retained the advantages of the avidin-biotin system: rapidity and good sensitivity.

A rapid and sensitive non-competitive avidin-biotin assay for lactoferrin.

We have developed an avidin-biotin assay for the detection of lactoferrin in human seminal plasma. We compared 5 modifications of this assay, and found the non-competitive avidin-biotin assay (NABA) to be the most sensitive. The detection limit of 3-step NABA was 0.2 or 0.1 ng lactoferrin/ml, depending whether avidin-biotin-peroxidase complex (ABC) or avidin-peroxidase was used. The intra-assay and inter-assay coefficients of variation for 3-step NABA were 6.2 and 8.5%, respectively. The lactoferrin levels of human seminal plasma samples measured by 3-step NABA and radioimmunoassay (RIA) showed good correlation (r = 0.96). The total performance time of 3-step NABA is flexible and the method can be modified for rapid (less than 1 h) or overnight assay according to need.

Sensitive immunometric assays for secretory peroxidase and myeloperoxidase in human saliva.

We have developed specific immunoassays for secretory peroxidase (SP) and for myeloperoxidase (MP) (polymorphonuclear leukocyte-derived peroxidase) in human saliva. Antibodies against SP and MP were produced using bovine milk lactoperoxidase (LP) and human MP as the immunogens, respectively. The methods developed are non-isotopic immunometric assays using biotinylated antibodies and avidin-enzyme conjugate. The detection limit was 0.1 ng/ml and the performance time less than 3 h for both assays. The determination ranges were 0.5-100 ng LP (SP)/ml and 0.5-200 ng MP/ml with intra- and interassay CVs of 4.3% and 15.6% for SP and 3.7% and 10.8% for MP, respectively. The mean analytical recoveries were 108.9% (SP) and 91.5% (MP). These assays correlated well (r = 0.849-0.871) with the colorimetric assays based on the oxidation of thiocyanate or chloride by peroxidases. However, compared to the colorimetric methods the new immunometric assays are much more sensitive and specific for salivary SP and MP. The assays are also more rapid since extensive dialysis to remove endogenous thiocyanate is not required.

Sucralfate mouth washing in the prevention of radiation-induced mucositis: a placebo-controlled double-blind randomized study.

PURPOSE: To evaluate the value of sucralfate mouth washings in prevention of radiation-induced mucositis. METHODS AND MATERIALS: Forty patients with head and neck cancer were randomized to use either sucralfate mouth washing 1 g six times daily during irradiation (n = 20) or to placebo washing (n = 20). Mouth washing was started at the beginning of radiation therapy and continued to the end of the therapy (7-10 weeks). Assessment of the degree of radiation mucositis and collection of stimulated saliva samples were done weekly during the therapy. Salivary lactoferrin and albumin, suggested markers for the degree of mucositis, were analyzed from stimulated whole saliva samples. RESULTS: All patients developed radiation-induced mucositis of varying degree after irradiation of about 30 Gy. No difference in the visually assessed degree of mucositis or oral pain reported by the patients was found between the study and the control groups. However, the patients treated with sucralfate used less anesthetic mouth washing and their salivary lactoferrin and albumin levels were lower. CONCLUSION: Although the trial produced no direct clinical evidence indicating that sucralfate mouth rinses prevent radiation-induced mucositis, the decrease in the salivary lactoferrin and albumin levels suggests that sucralfate has a slight protective effect on the oral mucosa.

Granulocyte macrophage-colony stimulating factor (GM-CSF) and sucralfate in prevention of radiation-induced mucositis: a prospective randomized study.

PURPOSE: To compare subcutaneously given molgramostim (GM-CSF) and sucralfate mouth washings to sucralfate mouth washings in prevention of radiation-induced mucositis. METHODS AND MATERIALS: Forty head and neck cancer patients were randomly assigned to use either GM-CSF and sucralfate (n = 20) or sucralfate alone (n = 20) during radiotherapy. Sucralfate was used as 1.0 g mouth washing 6 times daily after the first 10 Gy of radiotherapy, and 150-300 microg GM-CSF was given subcutaneously. The grade of radiation mucositis and blood cell counts were monitored weekly. Salivary lactoferrin was measured as a surrogate marker for oral mucositis. RESULTS: We found no significant difference between the molgramostim and the control groups in the oral mucositis grade, oral pain, use of analgesic drugs, weight loss, or survival. The median maximum neutrophil counts (median, 9.2 x 10(9)/L vs. 5.9 x 10(9)/L, p = 0.0005), eosinophil counts (median, 1.3 x 10(9)/L vs. 0.2 x 10(9)/L, p = 0.0004), and salivary lactoferrin concentrations were higher in patients who received GM-CSF. The most common toxicities in the GM-CSF plus sucralfate group were skin reactions at the GM-CSF injection site (65%), fever (30%), bone pain (25%), and nausea (15%), whereas the toxicity of sucralfate given alone was minimal. CONCLUSION: We found no evidence indicating that subcutaneously given GM-CSF reduces the severity of radiation-induced mucositis.

Specific detection of neuronal cell bodies: in situ hybridization with a biotin-labeled neurofilament cDNA probe.

We have used a biotinylated, 300-nucleotide cDNA probe which encodes the 68,000 MW neurofilament protein to detect neurofilament-specific mRNA in situ. The neurofilament message specifically demonstrates the neuronal cell bodies, in contrast to the usual antibody staining which detects their neurites. The hybridization is detected only in neuronal structures. Consequently, detection of the biotinylated neurofilament DNA probe by silver-intensified streptavidin-gold can be specifically used to identify neuronal cell bodies.

Immunocytochemical localization of human 5 alpha-reductase 2 with polyclonal antibodies in androgen target and non-target human tissues.

We studied the tissue distribution and cellular localization of 5 alpha-reductase 2, the human prostatic isoenzyme, in different human tissues, both cryostat-sectioned and paraffin-embedded. Polyclonal antibodies raised in rabbits against a native peptide (C-terminal amino acids 229-254) or synthetic peptides (amino acids 234-245), either as carrier-conjugated linear peptides or multiple antigen peptides (MAP), were assayed for specificity and sensitivity with Western blotting and an ELISA system. One antibody showing monospecificity on Western blots and in ELISA was used for immunohistochemical detection of the respective antigen in tissues from male and female subjects. Positive cells were found (with decreasing intensity) in inner epithelial sheath of hair follicles, pyramidal cells of the cerebral cortex, hepatocytes and bile duct cells, prostate epithelial cells, seminal vesicle epithelial cells, endothelial cells of small vessels, fat cells, fibrocytes of genital and extragenital organs, and smooth muscle cells of prostate and seminal vesicles. Some variation in the immunoreactivity of testis and ovary tissue was seen with different antibodies. 5 alpha-Reductase 2 is obviously not restricted to androgen target organs in the male, but is present in a large number of cells and tissues in both males and females.

Avidin and ovalbumin induction by progesterone in chicken oviduct detected by sensitive immunoenzymometric assays.

This study describes sensitive immunoenzymometric assays (IEMAs) for chicken avidin and ovalbumin, markers of cytodifferentiation and action of progesterone and oestrogen in the oviduct magnum mucosa. The determination range was 0.5-100 ng/ml and the detection limit 0.1 ng/ml in both IEMAs. The intra- and interassay coefficients of variation, measured from chicken tissue supernatants, averaged below 6 and 10% respectively. IEMAs correlated well with the radioimmunoassays for avidin and ovalbumin previously developed in our laboratory, and with the widely used [14C]biotin-binding method for avidin. Using an IEMA, we found avidin induction with low concentrations of progesterone in the differentiated oviduct of oestrogen-pretreated chicks. The induction has not been detected previously by less sensitive methods. Avidin was induced by all given doses of progesterone (0.2-200 mg/kg in vivo for 24 h after a short oestrogen treatment), the response being dose-dependent at doses of 0.2-20 mg progesterone/kg body weight, the maximum avidin production being about 70 micrograms/g tissue. Ovalbumin was induced at doses of 2-200 mg progesterone/kg body weight without variations in the responses, being about 35 mg/g. The mean content of avidin in the oviduct of laying hens was 58.1 micrograms/g, and of ovalbumin 74.9 mg/g. Minimal traces of avidin and ovalbumin were found in the oviduct after hatching (0.3 and 5 micrograms/g respectively); however, progesterone did not have an effect on this expression. Sensitivity, rapidity and practicability, together with non-radioactivity, are the main advantages of the present IEMAs for chicken avidin and ovalbumin.

Development and evaluation of an immunoenzymometric assay for the chicken progesterone receptor.

A specific and sensitive immunoenzymometric assay (IEMA) was developed for measuring the quantity of chicken progesterone receptor (PR) in tissue cytosol. The assay uses two monoclonal antibodies to the PR. One is used to capture the PR. The second (labelled with biotin) reacts first with the captured receptor and subsequently with avidin-labelled horseradish peroxidase to provide an enzymatic end-point. The method has a determination range from 0.3 to 60 pmol/l. Intra- and interassay coefficients of variation were 3.7% and 9.0% respectively. The assay can be performed with equal results as a rapid (3 h) or an overnight procedure. The IEMA is convenient, especially for signal measurement and the calculation of results. No ultracentrifugation of samples is needed, since the IEMA can be performed on low-speed cytosol samples. Assay results correlated well (r = 0.927) with those obtained by the conventional ligand-binding assay used in our laboratory. Similar results were obtained with the IEMA and the ligand-binding assay after exposure of cytosol samples to increased temperatures: at 20 degrees C the PR remained stable for the 4-h period examined, whereas at 37 degrees C almost complete degradation of the PR was observed in 30 min. Being more than 100 times as sensitive as the ligand-binding assay, the IEMA enabled the quantification of PR for the first time in such tissues as the bursa and small intestine even of immature animals.

Characterization of the estrogen-sensitive cells expressing progesterone receptor in the bursa of Fabricius.

Cells expressing the progesterone receptor (PR) in the bursa of Fabricius (BF) were studied with immunohistochemistry at light-microscopic level, with immunoelectron microscopy (immuno-EM) and with non-specific esterase histochemistry. The antibody (IgG-RB) directed to the B component of the chick oviduct progesterone receptor was shown by immunoblotting to be specific for the PR and to recognize the PR also in the bursa. Two cell types in the BF contain the PR: stromal cells in the interfollicular-subepithelial area and smooth muscle cells lining the BF. The PR was localized in the nuclei of these cells. The bursal epithelium and the cells inside the follicles were not stained for PR. Electron microscopically the immunoreaction precipitate was localized on condensed heterochromatin and on dispersed euchromatin. The cells expressing the PR resembled electron microscopically fibroblasts. Their cytoplasm was rich in rough endoplasmic reticulum indicating active protein synthesis. By non-specific esterase histochemistry we showed that the PR-containing cells were not macrophages, which are morphologically indistinguishable from stromal cells. In the bursae of young untreated chicks the PR was not seen, but was inducible by estradiol treatment and was spontaneously expressed after the onset of sexual maturation. It is concluded that both the stromal fibroblasts and the smooth muscle cells in the BF are estrogen and progesterone sensitive. The expression of PR after the onset of sexual maturation indicates that the BF is directly affected by sexual maturation-associated factors. We suggest that estrogen and progesterone participate in tissue remodelling during bursal involution via the stromal cells and may affect bursal functions via the smooth muscle cells.

Subcellular location of unoccupied and occupied glucocorticoid receptor by a new immunohistochemical technique.

Recent immunohistochemical studies suggest that the unoccupied glucocorticoid receptor (GR) is cytoplasmic and that the ligand causes its translocation into the target cell nucleus. The subcellular location of GR is especially interesting in that other members of the steroid receptor superfamily appear to be nuclear. The intracellular distribution of GR was studied immunohistochemically using a new freeze-drying and vapor fixation method which eliminates the protein diffusion and redistribution possibly caused by liquid fixation techniques. We used two monoclonal antibodies against rat liver GR. Dried samples of the adrenalectomized rat brain and uterus were fixed in p-benzoquinone vapor for 3 h at 60 degrees C and embedded in paraffin. Sections were stained with a biotinylated mouse monoclonal GR antibody using the avidin-biotin-peroxidase complex. Both unoccupied and occupied GR were found in the nucleus of the target cells, fibroblasts in the uterus and nerve cells in the cortex of the brain. The staining was saturated with the cytosol of cos cells transfected with GR. No cytoplasmic staining was seen even 2 days after adrenalectomy. In conclusion we propose that GR is also located in the nucleus independently of occupation.

Inducibility of the avidin gene by progesterone is suppressed during estrogen-induced cytodifferentiation.

We have studied epithelial differentiation of the chick oviduct as induced by diethylstilbestrol (DES) and 17 beta-estradiol (E2). The proportion of goblet cells in the oviduct was slightly higher after E2 than after DES treatment. Also avidin induction by progesterone was stronger following DES than E2 priming. In the estrogen pretreated oviduct epithelium, avidin expression was induced by progesterone in the surface epithelial cells, protodifferentiated gland cells and tubular gland cells, but not in goblet cells. During prolonged estrogen treatment, however, the inducibility of avidin by progesterone ceased in tubular gland cells but not in surface epithelial cells. The estrogen action on the expression of avidin could be explained by estrogen-induced terminal differentiation of the epithelial gland cells or by a direct effect of estrogen on the progesterone action, for instance interaction of estrogen receptor and progesterone receptor in the regulation of transcription.

Characterization of human 1,25-dihydroxyvitamin D3 receptor anti-peptide antibodies.

Rabbit and chicken antibodies were raised against two peptides synthesized according to the structure of human 1,25-dihydroxyvitamin D3 receptor (hVDR): rabbit alpha hVDR-103 against the N-terminal amino acids 5-18 and alpha hVDR-104 against the amino acids 172-186 in the hinge region and chicken alpha hVDR-cab11 against the amino acids 172-186, respectively. The specificity of the antibodies was tested by peptide saturation, SDS-PAGE immunoblotting, gel shift assay and sucrose gradient centrifugation. Immunoblotting of a soluble extract (cytosol) from osteosarcoma cell line MG-63 showed a single band with an M(r) of about 48,000 and human intestine cytosol a broad band (50-63,000) for both antibodies. The antibodies recognized activated (3.2S) hVDR by shifting the centrifugation sedimentation profile to 5-6S. The antibodies showed nuclear immunostaining of unoccupied VDR in human osteosarcoma cells MG-63, U2-Os and SaOs-2. The immunoreaction could be saturated with the corresponding synthetic peptide. In immunoblot alpha hVDR-103 reacted with human and rat VDR, whereas alpha hVDR-104 recognized human VDR only. Similarly in immunohistochemistry, alpha hVDR-103 showed staining with hVDR and rVDR, whereas alpha hVDR-104 reacted only with hVDR. All antibodies recognized the native hVDR as verified with sucrose gradient centrifugation or immunoprecipitation but only alpha hVDR-103 and alpha hVDR-cab11 in gel shift assay of hVDR associated with the vitamin D-responsive element of human osteocalcin gene promoter.

Vitamin D and prostate cancer.

Our recent epidemiological study (Ahonen et al., Cancer Causes Control 11(2000) (847-852)) suggests that vitamin D deficiency may increase the risk of initiation and progression of prostate cancer. The nested case-control study was based on a 13-year follow-up of about 19000 middle-aged men free of clinically verified prostate cancer. More than one-half of the serum samples had 25OH-vitamin D (25-VD) levels below 50 nmol/l, suggesting VD deficiency. Prostate cancer risk was highest among the group of younger men (40-51 years) with low serum 25-VD, whereas low serum 25-VD appeared not to increase the risk of prostate cancer in older men (>51 years). This suggests that VD has a protective role against prostate cancer only before the andropause, when serum androgen concentrations are higher. The lowest 25-VD concentrations in the younger men were associated with more aggressive prostate cancer. Furthermore, the high 25-VD levels delayed the appearance of clinically verified prostate cancer by 1.8 years. Since these results suggest that vitamin D has a protective role against prostate cancer, we tried to determine whether full spectrum lighting (FSL) during working hours could increase serum 25-VD concentrations. After 1-month exposure, there was no significant increase in the serum 25-VD level, although there was a bias towards slightly increasing values in the test group as opposed to decreasing values in controls. There was no significant change in the skin urocanic acid production. The possibility to use FSL in cancer prevention is discussed. In order to clarify the mechanism of VD action on cell proliferation and differentiation, we performed studies with the rat and human prostates as well prostate cancer cell lines. It is possible that 25-VD may have a direct role in the host anticancer defence activity, but the metabolism of vitamin D in the prostate may also play an important role in its action. We raised antibodies against human 1alpha-hydroxylase and 24-hydroxylase. Our preliminary results suggest that vitamin D is actively metabolised in the prostate. Vitamin D appears to upregulate androgen receptor expression, whereas androgens seem to upregulate vitamin D receptor (VDR). This may at least partially explain the androgen dependence of VD action. VD alone or administered with androgen causes a suppression of epithelial cell proliferation. VD can activate mitogen-activated kinases, erk-1 and erk-2, within minutes and p38 within hours. Also, auto/paracrine regulation might be involved, since keratinocyte growth factor (mRNA and protein) was clearly induced by VD. Based on these studies, a putative model for VD action on cell proliferation and differentiation is presented.

Spontaneous luteinizing hormone surge and cleavage of in vitro fertilized embryos.

The importance of monitoring luteinizing hormone (LH) secretion during gonadotropin stimulation remains controversial. In the present study, the authors evaluated the occurrence of spontaneous LH surges in 170 cycles stimulated by clomiphene citrate and human menopausal gonadotropin, and correlated the success rate of embryo cleavage to the time interval between the occurrence of the LH surge peak value and the time of human chorionic gonadotropin (hCG) administration. LH was quantitated from urine by an avidin-biotin enzyme immunoassay. The results indicated that a spontaneous LH surge occurred in 18% of the cycles. The number of oocytes recovered was not affected by the occurrence of a spontaneous LH surge. In 12% of all cases, the spontaneous LH surge occurred less than 12 hours before the administration of hCG, and in these cases embryo cleavage was not reduced. In 6% of all cases, the spontaneous LH surge occurred over 12 hours before hCG administration, and in these cases embryo cleavage was reduced significantly.

Norplant implants: the mechanism of contraceptive action.

OBJECTIVE: To determine if fertilization occurs unnoticed among Norplant users who are ovulatory. DESIGN: Serial blood samples were obtained during 1 month from sexually active Norplant users experiencing regular menstrual bleeding patterns and a control group of noncontracepting women trying to conceive. The sequential blood samples were assayed for the presence of human chorionic gonadotropin (hCG). SETTING: All samples were obtained from women receiving contraceptive service and health care at the Center for Research and Services in Human Reproduction and Contraception, Santo Domingo, The Dominican Republic. Assays for hCG were performed at the Department of Biomedical Sciences, University of Tampere, Finland. PATIENTS, PARTICIPANTS: A total of 32 women using Norplant implants were enrolled in the treatment group, and 20 women of proven fertility who were attempting to conceive served as a control group. INTERVENTIONS: Duration of Norplant use was as follows: 4 in the 2nd year of use, 13 in the 3rd year, 11 in the 4th year, 3 in the 5th year, and 1 in the 7th year. MAIN OUTCOME MEASURE: The determination of pregnancy was based on the presence of hCG in the luteal phase, using a sensitive and specific immunoenzymatic assay that can detect dimeric hCG as early as 7 days after ovulation. RESULTS: Nine pregnancies were detected. All were in the control group trying to conceive. Six of these advanced to clinical pregnancies, and three did not proceed beyond the next expected menses. None of the Norplant users had evidence of hCG production, whether the observed cycles were anovulatory or ovulatory. The probability of finding no pregnancies in the ovulatory months at risk among Norplant users is between 1 in 50 and 1 in 150,000. The null hypothesis that Norplant users conceive at a natural rate can be rejected at the 0.05 level. CONCLUSION: Interruption of early pregnancy (menstrual abortion) does not play a role in the mechanism of action of Norplant contraceptive implants.

PIP: This study sought to determine if fertilization can occur unnoticed among Norplant users who are ovulatory. Serial blood samples were obtained during a 1-month period from women receiving contraceptive service and healthcare at the Center for Research and Services in Human Reproduction and Contraception, Santo Domingo, The Dominican Republic. These women were all sexually active Norplant users who experienced regular menstrual bleeding patterns, and their sequential blood samples were assayed for the presence of human chorionic gonadotropin (hCG) at the Department of Biomedical Sciences at the University of Tampere, Finland. There was also a control group of 20 noncontracepting women included who were trying to conceive. Duration of Norplant was as follows: 4 in the 2nd year of use, 13 in the 3rd year, 11 in the 4th, 3 in the 5th year, and 1 in the 7th year. Pregnancy determination was based on the presence of hCG in the luteal phase by use of a sensitive and specific immunoenzymatic assay that can detect dimeric hCG as early as 7 days postovulation. 9 pregnancies were detected, all in the control group who were trying to conceive. 6 of these advanced to clinical pregnancies and 3 terminated spontaneously at the next menstrual period. None of the Norplant users evidenced and hCG production, whether or not the observed cycles were ovulatory. The probability of finding no pregnancies in the ovulatory months at risk among Norplant users is between 1/50 and 1/150,00. The null hypotheses that Norplant users conceive at a natural rate can be rejected at the 0.05 level. Thus, interruption of early pregnancy (menstrual abortion) does not play a role in the mechanism of action of Norplant contraceptive implants.

Longitudinal analysis of the association of human salivary antimicrobial agents with caries increment and cariogenic micro-organisms: a two-year cohort study.

Previous studies of the possible associations of salivary antimicrobial agents with dental caries have given controversial results, obviously mainly because almost all studies have been cross-sectional. Our aim was to find out, in a two-year longitudinal follow-up study, the associations among selected salivary non-immune and immune antimicrobial variables, cariogenic bacteria, and caries increment. The study population was comprised of 63 subjects, all of whom had their 13th birthday during the first study year. In addition to a comprehensive dental examination at baseline and after 2 yrs, paraffin-stimulated whole saliva samples were collected in a standardized way at six-month intervals. Saliva samples were analyzed for flow rate, buffer effect, lysozyme, lactoferrin, total peroxidase activity, hypothiocyanite, thiocyanate, agglutination rate, and total and specific anti-S. mutans IgA and IgG, as well as for numbers of total and mutans streptococci, lactobacilli, and total anaerobic bacteria. Cluster analysis and Spearman-Rank correlation coefficients were used to explore possible associations between and among the studied variables. During the two-year period, a statistically significant increase was observed in flow rate, thiocyanate, agglutination rate, anti-S. mutans IgA antibodies, lactobacilli, and total anaerobes, whereas lysozyme, lactoferrin, and total and anti-S. mutans IgG antibodies declined significantly. Based on various analyses, it can be concluded that, at baseline, total IgG and hypothiocyanite had an inverse relationship with subsequent two-year caries increment, anti-S. mutans IgG antibodies increased with caries development, and mutans streptococci and lactobacilli correlated positively with both baseline caries and caries increment. Total anaerobic microflora was consistently more abundant among caries-free individuals. In spite of the above associations, we conclude that none of the single antimicrobial agents as such has sufficiently strong power to have diagnostic significance in vivo with respect to future caries.

Antimicrobial factors in saliva: ontogeny and relation to oral health.

Antimicrobial agents (antibody and non-antibody) present in human saliva protect oral tissues by a variety of mechanisms, such as prevention of bacterial adhesion, agglutination of micro-organisms, and inhibition of multiplication and metabolism. However, studies in which the concentrations of various salivary antimicrobial agents have been correlated to the presence and severity of oral diseases--of dental caries, in particular--have produced controversial data, and it seems evident, also on the basis of the present study, that no single salivary antimicrobial factor (except flow rate) affects oral health to a significant degree. In the present study, we report the levels of some selected salivary antimicrobial agents in predentate and dentate human infants, with a comparison to the levels found in young adults' saliva. Salivary lysozyme, peroxidase, and hypothiocyanite concentrations were already at the adult level at the time when the primary teeth erupt, whereas immunoglobulin (IgA, IgG, and IgM), lactoferrin, myeloperoxidase, and thiocyanate concentrations were significantly lower in children than in adults. Dentate children had more IgG, thiocyanate, and protein in whole saliva than did predentate children.

Immunoglobulins and innate antimicrobial factors in whole saliva of patients with insulin-dependent diabetes mellitus.

We analyzed the flow rate and composition of paraffin-stimulated whole saliva samples from 35 adult diabetic patients and their age- and sex-matched, non-diabetic, clinically healthy controls. All patients had insulin-dependent diabetes (IDDM) with a mean (+/- S.D.) duration of 14.0 +/- 9.1 years. The saliva analysis included the quantitation of total protein, amylase, immunoglobulins (isotypes A, G, and M), and the non-antibody, innate antimicrobial factors (lysozyme, lactoferrin, salivary peroxidase, myeloperoxidase, thiocyanate, and hypothiocyanite). The whole saliva samples from diabetic patients had significantly higher amounts of IgA (p less than 0.001) and IgG (p less than 0.05) than did the controls. No differences between the study groups were observed in flow rate, protein content, amylase activity, or IgM. The levels of innate defense factors were similar in both study groups except for salivary peroxidase, which was higher (p less than 0.02) among diabetics than among controls. Our results indicate that the antimicrobial defense capacity of whole saliva is not impaired in diabetic patients.