Conditions influencing the in vitro antifungal activity of lactoferrin combined with antimycotics against clinical isolates of Candida. Impact on the development of buccal preparations of lactoferrin.

Lactoferrin, an iron-binding glycoprotein, is a potential agent for the treatment of oropharyngeal Candidiasis. The aim of the present study was to test the capability of lactoferrin, combined or not combined with conventional antifungal agents, to inhibit the growth of different Candida species under various experimental conditions to be of guidance in the development of a suitable pharmaceutical formulation containing lactoferrin. The anti-Candida activities of lactoferrin were considerably higher using RPMI instead of SLM as assay medium. They were moreover increased by raising the medium pH from 5.6 to 7.5. With the 'standard' antifungal agent fluconazole similar results were found as for lactoferrin, but the medium type and pH did not affect MIC values of amphotericin B. The addition of saliva to medium did not reduce the antifungal activities of the individual compounds. Synergistic inhibitory effects on Candida growth were found for combinations of lactoferrin and fluconazole or amphotericin B, irrespective of the medium type and pH, or the addition of saliva. This indicates that for treatment of oral Candidiasis a formulation containing lactoferrin seems appropriate; results may be optimized if the formulation is provided with buffer capacity to attain pH 7.5 in the mucosal fluid. The synergistic effects between lactoferrin and 'standard' antifungals indicate that combinations should be considered in such a formulation.

Synergistic fungistatic effects of lactoferrin in combination with antifungal drugs against clinical Candida isolates.

Because of the rising incidence of failures in the treatment of oropharyngeal candidosis in the case of severely immunosuppressed patients (mostly human immunodeficiency virus [HIV]-infected patients), there is need for the development of new, more effective agents and/or compounds that support the activity of the common antifungal agents. Since lactoferrin is one of the nonspecific host defense factors present in saliva that exhibit antifungal activity, we studied the antifungal effects of human, bovine, and iron-depleted lactoferrin in combination with fluconazole, amphotericin B, and 5-fluorocytosine in vitro against clinical isolates of Candida species. Distinct antifungal activities of lactoferrin were observed against clinical isolates of Candida. The MICs generally were determined to be in the range of 0.5 to 100 mg. ml(-1). Interestingly, in the combination experiments we observed pronounced cooperative activity against the growth of Candida by using lactoferrin and the three antifungals tested. Only in a limited concentration range was minor antagonism detected. The use of lactoferrin and fluconazole appeared to be the most successful combination. Significant reductions in the minimal effective concentrations of fluconazole were found when it was combined with a relatively low lactoferrin concentration (1 mg/ml). Such combinations still resulted in complete growth inhibition, while synergy of up to 50% against several Candida species was observed. It is concluded that the combined use of lactoferrin and antifungals against severe infections with Candida is an attractive therapeutic option. Since fluconazole-resistant Candida species have frequently been reported, especially in HIV-infected patients, the addition of lactoferrin to the existing fluconazole therapy could postpone the occurrence of species resistance against fluconazole. Clinical studies to further elucidate the potential utility of this combination therapy have been initiated.

Relative frequencies of cystic fibrosis mutations in The Netherlands as an illustration of significant regional variation in a small country.

Cystic fibrosis (CF) is one of the most common autosomal recessive disorders in white populations. Significant regional differences in CF mutations among affected individuals have been reported. We have studied the geographic distribution of the relative frequencies of the three most common Dutch CF mutations, deltaF508, A455E, and G542X, by analyzing data on area of residence of CF patients. Significantly higher relative frequencies of the A455E mutation and the G542X mutation were observed in the South-West and the South-East, respectively. A uniform distribution of relative frequencies was found for the deltaF508 mutation. The results of our study show that, even in a small country such as The Netherlands, certain CF mutations may be more common in one region than in another.

Human uteroglobin gene: structure, subchromosomal localization, and polymorphism.

Human uteroglobin (hUG) or Clara cell 10-kD protein (cc10 kDa) is a steroid-dependent, immunomodulatory, cytokine-like protein. It is secreted by mucosal epithelial cells of all vertebrates studied. The cDNA encoding hUG and the 5' promoter region of the gene have been characterized previously. Here, we report that the structure of the entire hUG gene is virtually identical to those of rabbit, rat, and mouse. It is localized on human chromosome 11q12.3-13.1, a region in which several important candidate disease genes have been mapped by linkage analyses. Our data indicate that candidate genes for atopic (allergic) asthma and Best's vitelliform macular dystrophy are in closest proximity to the hUG gene. To determine whether hUG gene mutation may be involved in the pathogenesis of these diseases, we studied two isolated groups of patients, each afflicted with either atopy or Best's disease, respectively. We detected a single base-pair change in the hUG gene in Best's disease patients and normal controls but no such change was detected in atopy patients. This alteration in hUG gene-sequence in Best disease family appears to be a polymorphism. Although the results of our investigation did not uncover mutations in hUG gene that could be causally related to the pathogenesis of either of these diseases, its conservation throughout vertebrate phyla implies that this gene is of physiological importance. Moreover, the close proximity of this gene to several candidate disease genes makes it an important chromosomal marker in cloning and characterization of those genes.

Prevalence of delta F508 cystic fibrosis carriers in The Netherlands: logistic regression on sex, age, region of residence and number of offspring.

An average cystic fibrosis (CF) carrier frequency of 1 in 25 in Europe is cited in numerous reports, although a great variability in estimated prevalences has been found in different European populations. The estimates of these frequencies were based on numbers of CF patients before identification of the gene in 1989. Here we report the results of a study to determine the carrier frequency of the delta F508 mutation in The Netherlands by analyzing mouthwashes and matched blood samples from 11654 blood donors all over the country. We analyzed possible relationships between a number of theoretically explanatory variables and the delta F508 carrier frequency by means of univariate and multivariate logistic regression. These variables were: distance of the blood banks from the northeastern part of the country (distance); whether the blood donors knew that we were looking for a CF mutation; sex and age of the donor; and number of children of the donor (family size). We detected a delta F508 carrier frequency of 1 in 42 (95% CI 1/37-1/47) in The Netherlands. If we assume that the relative frequency of the delta F508 mutation among carriers and patients is comparable in The Netherlands, this gives an estimated overall CF carrier frequency of 1 in 32 (95% CI 1/28-1/36), significantly less than 1 in 25. The univariate logistic regression analysis of the effects of the explanatory variables on the carrier frequency revealed no significant relationships, except for an increase in carrier frequency with increasing distance from the northeastern region. In the multivariate analysis with all five independent variables, distance, age and family size were significantly related to the carrier frequency, but sex and CF information were not. There was a significant interaction between age and family size. In our final model, distance, age and family size were positively related to the carrier frequency, while the interaction of age with family size showed a negative relation. These results confirm that there is a gradient in gene frequency with low frequencies in the northeastern part of the country and high frequencies in the southern part. They also suggest a relation of age and family size with carrier frequency. This relation, however, is too complex to be explained by heterozygote advantage.

Haplotype identity between individuals who share a CFTR mutation allele "identical by descent": demonstration of the usefulness of the haplotype-sharing concept for gene mapping in real populations.

Cystic fibrosis (CF) patients with the A455E mutation, in both the French Canadian and the Dutch population, share a common haplotype over distances of up to 25 cM. French Canadian patients with the 621+1G-->T mutation share a common haplotype of more than 14 cM. In contrast, haplotypes containing the delta F508 mutation show haplotype identity over a much shorter genomic distance within and between populations, probably because of the multiple introduction of this most common mutation. Haplotype analysis for specific mutations in CF or in other recessive diseases can be used as a model for studying the occurrence of genetic drift conditional on gene frequencies. Moreover, from our results, it can be inferred that analysis of shared haplotypes is a suitable method for genetic mapping in general.

Prevalence of carriers of the most common medium-chain acyl-CoA dehydrogenase (MCAD) deficiency mutation (G985A) in The Netherlands.

The G985A mutation represents about 90% of all medium-chain acyl-CoA dehydrogenase (MCAD) allele mutations that cause the clinical symptoms of MCAD deficiency. The prevalence of carriers varies between different European populations, with high frequencies in the northwestern part of Europe. To determine the prevalence of MCAD carriers with the G985A mutation in The Netherlands, we collected 6195 Guthrie cards of newborns. Mutation detection was performed with the polymerase chain reaction (PCR), in which a NcoI restriction site was created in the presence of a G985A mutation in the PCR product, followed by NcoI digestion, and gel electrophoresis. We detected a G985A carrier frequency of 1 in 59 (95% CI 1/50-1/73) in The Netherlands. The total prevalence of carriers was estimated to be 1 in 55 (95% CI 1/46- 1/68), based on a relative G985A frequency of 94% in The Netherlands.

Validation of the determination of deltaF508 mutations of the cystic fibrosis gene in over 11 000 mouthwashes.

Mouthwashes can be used as a DNA resource for mutation detection and, because collection and DNA isolation is simple and cheap, they could in particular, be used for large numbers of samples. To determine the failure rate (the proportion of mouth samples in which no PCR product was obtained) and the specificity of buccal epithelial cell mutation detection in large numbers of samples, we collected mouthwashes and blood samples from 11 413 blood donors and tested the mouthwashes for the deltaF508 mutation, which has an estimated frequency Of 75% among cystic fibrosis chromosomes in The Netherlands. Blood samples were tested for the deltaF508 mutations only if the mutation was identified in the mouthwash or in the case of a failure to obtain PCR products. The sensitivity of the test was determined in mouthwashes of 75 deltaF508 carriers known from earlier family studies. These samples were offered blindly between the mouthwashes of the blood donors. Both specificity and sensitivity of the mouthwash procedure were 100%. The overall failure rate was 5.6%. This large figure was caused mainly by insufficient rinsing of the mouth in one particular blood bank. Exclusion of the results of this blood bank reduced the failure rate to 1.8%. Our results also confirmed that for a large number of samples the mouthwash procedure is suitable for mutation detection and, with proper instructions, can be used in community screening.

Number and sex of offspring of delta F508 carriers outside cystic fibrosis families.

Number and sex of offspring were determined in a group of 7,841 randomly selected blood donors who were screened for the delta F508 mutation. We did not find any evidence for differences in number or sex ratio of offspring between delta F508 carriers and non-carriers.

Electron spin resonance in zero magnetic field of the reaction center triplet of photosynthetic bacteria.

The decay rates kx, ky, kz of the individual spin levels of the light-induced triplet state have been accurately measured by the zero-field resonance technique under conditions of very low light intensity and a microwave sweep rate of 2.5 MHz/microseconds, which is in excess of that commonly used in optical detection magnetic resonance experiments. The rates ku found correspond well with those previously determined under somewhat different conditions (Hoff, A.J. (1976) Biochim. Biophys. Acta 440, 765--771) and with those inferred from the decay at 4.2 degrees K of the triplet-triplet absorption after picosecond excitation (Parson, W.W. and Monger, T.G. (1977) Brookhaven Symp. Biology 28, 195--212). Thus there seems no reason to doubt that PR corresponds to the triplet state detected by ESR. In a recent publication Clarke and Connors (Clarke, R.H. and Conners, R.E. (1976) Chem. Phys. Lett. 42, 69--72) published values of the rates ku which differ substantially from ours and which lead to a mean lifetime in excess of that of PR. We show that erroneous rates are obtained when the microwave sweep rate is not made fast relative to the decay of the individual spin levels. Zero-field splitting parameters for a member of photosynthetic bacteria have been measured with an accuracy of better than 0.4% for D and 1% for E. The enhanced precision as compared to conventional ESR allows one to discriminate between species of one family. Deuteration reduces the ku values by a factor of about 2, with little spin selectivity. This effect is much larger than previously observed for chlorophyll a. The present results explain the decrease in fluorescence intensity observed on microwave saturation in zero-field optical detection magnetic resonance experiments, and they also show that the simple exciton model is inadequate to derive the geometry of the reaction center dimer from the observed zerofield splitting and decay rates.