[Toxicological risk assessment using the example of potential contact sensitization to resorcinol]. / Toxikologische Risikobewertung am Beispiel einer möglichen Kontaktsensibilisierung auf Resorcinol.

Resorcinol is a frequently used hair dye, whose quantitative risk assessment (QRA) for hair color products is presented in this review as an example to assess its skin sensitization risk after topical application. Its purpose is to determine the maximum concentration that can be used without expecting skin sensitization to occur. The focus is to prevent the de novo development of a contact allergy. Epidemiological data which are provided via dermatological surveillance, e.g., by the IVDK (Information Network of Departments of Dermatology) in Germany, are an important source of information that help to assess the quality and the effectivity of the QRA.

Cross-elicitation responses to 2-methoxymethyl-p-phenylenediamine in p-phenylenediamine highly allergic volunteers using allergy alert test: the Italian experience.

Summary: Background. Allergic contact dermatitis after exposure to p-phenylenediamine (PPD)-containing hair dye products is a common and important clinical problem. Because there is a high rate of cross-elicitation of allergic contact dermatitis to other important hair dye products (such as p-toluene diamine [PTD] and other aminophenol hair dyes) in PPD allergic patients, safer alternative dyes with excellent hair coloring options are needed. We studied 2-methoxy methyl-PPD (Me-PPD), a chemical derivative of PPD for tolerance versus cross-elicitation in a cohort of eight PPD-allergic volunteers. Objective. To study tolerance to Me-PPD in a PPD highly allergic Italian cohort. Methods. Eight volunteers with a history of contact dermatitis to hair dyes or other PPD-containing chemicals and positive patch tests to 1% PPD in petrolatum, were recruited to study their immediate and delayed skin reactivity to PPD, vehicle control and 2-methoxy-methyl-PPD (Me-PPD), using the allergy alert test (simulating hair dyeing conditions) on volar forearm skin. This is a short-contact open patch test. Results. All eight volunteers reacted to PPD allergy alert test (100%); none reacted to vehicle (0%), and seven of eight reacted to Me-PPD allergy alert test (88%). However, in those seven volunteers who exhibited cross-elicitation to Me-PPD, their aggregate skin test reactivity to Me-PPD was significantly less than that of PPD (figure 3, p minore 0.0062, highly significant, paired two-tailed, students t test). Conclusions. Me-PPD may offer a safer alternative for PPD-allergic patients with an absent or reduced elicitation response in the allergy alert test simulating hair dye use conditions. Even patients with strong patch test reactions, with appropriate selection by allergy alert test and counselling, may be able to tolerate hair dyeing with Me-PPD containing products.

[Lymphadenitis and systemic lupus erythematosus]. / Lymphadenitis und systemischer Lupus erythematodes.

A 25-year-old Caucasian female patient presented with fever and cervical lymphadenopathy. Laboratory findings showed elevated signs of inflammation, elevated ANA titer and strongly positive anti-dsDNA antibodies. The histopathology of the lymph nodes revealed distinct features of Kikuchi-Fujimoto disease, a benign, self-limiting lymphadenopathy that typically affects young Asian females. In the literature a coincidental occurrence of Kikuchi-Fujimoto disease and systemic lupus erythematosus (SLE) is well documented. We hypothesized a simultaneous occurrence of both diseases because of the typical antibodies and arthralgia.

Continuous usage of a hair dye product containing 2-methoxymethyl-para-phenylenediamine by hair-dye-allergic individuals.

BACKGROUND: Despite a positive patch test reaction to para-phenylenediamine (PPD) and/or toluene-2,5-diamine (PTD), many people attempt to continue dyeing their hair with products containing PPD or its derivatives. OBJECTIVES: Investigation of elicitation reactions among PPD/PTD-allergic individuals to hair dye products containing the less sensitizing PPD derivative 2-methoxymethyl (ME)-PPD. METHODS: Elicitation reactions were studied in 43 PPD/PTD-allergic individuals by a 45-min pretest with an ME-PPD-containing hair dye on their forearm. Upon a negative result this was followed by exposure to subsequent hair colour treatment(s). RESULTS: Overall, 38 of 43 PPD/PTD-allergic individuals did not develop an elicitation reaction during the pretest with ME-PPD-containing hair dye products, and were eligible for subsequent hair colour treatments. Of these 38 PPD/PTD-allergic individuals, 29 tolerated subsequent hair dyeing with ME-PPD-containing hair dye products, while seven showed mild and two showed moderate/marked allergic reactions upon the first hair colour treatment. CONCLUSIONS: Hair dye products with the less sensitizing ME-PPD were tolerated by 29 of 43 (67%) PPD/PTD-allergic individuals throughout continued hair dyeing with an average of nine treatments per year. Five individuals reacted upon pretesting, while only mild-to-moderate/marked skin reactions occurred upon hair dyeing in nine individuals who were not identified by the pretest. To our knowledge this is the first study among PPD/PTD-allergic individuals indicating that a negative 45-min pretest with a hair dye product helps to avoid severe allergic reactions.

Cross-elicitation responses to 2-methoxymethyl-p-phenylenediamine under hair dye use conditions in p-phenylenediamine-allergic individuals.

BACKGROUND: The factors influencing elicitation responses in individuals allergic to p-phenylenediamine (PPD) in hair dyes are not well understood. OBJECTIVES: Investigation of the elicitation response to the new, less-sensitizing PPD alternative 2-methoxymethyl-p-phenylenediamine (ME-PPD) under simulated hair dye use conditions. METHODS: The cross-elicitation response to ME-PPD (2% in a hair dye test product for 30 min on forearm then rinsing) was analysed at days 2 and 3 in 30 PPD-allergic individuals with diagnostic patch test grades +, ++ or +++ according to the classification of the International Contact Dermatitis Research Group. RESULTS: Cross-reactivity to the ME-PPD-containing hair dye test product was elicited in nine of 30 subjects (30%), while 70% were negative. Cross-reactivity was elicited in two of four cases with grade +++, three of 10 with grade ++ and four of 16 with grade +. Under identical conditions, PPD was previously found to elicit a response in 21 of 27 PPD-allergic individuals. In 18 of these 21 individuals, either the strength of the cross-elicitation response to ME-PPD was decreased or no response occurred. CONCLUSIONS: Under simulated hair dye use conditions, a significantly lower degree of cross-elicitation to ME-PPD (30%) was observed than previously reported for PPD (32 of 38, 84%). Additionally, a decreased cross-elicitation strength was observed across all three patch test grades, likely reflecting the reduced skin-sensitization properties of ME-PPD. Consequently, careful dermatological evaluation is required to assess cross-reactivity to ME-PPD in patients allergic to hair dyes.

Assessment of the elicitation response in subjects weakly sensitized to p-phenylenediamine.

BACKGROUND: A 30-min application of a hair dye product containing 2% p-phenylenediamine (PPD) to subjects diagnostically graded +, showed that 12 of 18 reacted; eight of 18 with a true + and four of 18 with a doubtful (?+) response, whereas six of 18 did not react at all. In vitro skin-binding experiments showed that for diagnostic patch test conditions the measured exposure level (MEL) is more than 10-fold higher than the MEL for hair dyeing conditions. OBJECTIVE: To further analyse the limited elicitation response of the diagnostically + graded subjects to a PPD hair dye product, under standardized test conditions mimicking product usage, by varying exposure time and dose. METHODS: A hair dye model formulation containing 2% PPD, applied for 30, 45 and 60 min and a diagnostic PPD TRUE test(®) were applied to assess elicitation responses to increasing PPD exposure levels. Grading was performed according to International Contact Dermatitis Research Group guidelines. RESULTS: Six subjects were available for this follow-up study. One of six subjects responded with a + elicitation response to the hair dye model applied for 60 min. Four of the five remaining subjects elicited a + response to the PPD TRUE test(®) applied subsequently, while one of five responded doubtfully. CONCLUSIONS: Increasing the PPD exposure time twofold--resulting in a 5-6% increase of sensitivity of this hair dye model test--or further extending the exposure time 48-fold, was found sufficient to increase the MEL above the thresholds needed to elicit individuals with a + diagnostic PPD patch test who did not react to typical hair dye use conditions with a MEL of about 6·8 µg cm⁻². This analysis confirms that consideration of the MEL is a useful tool to better characterize thresholds of elicitation than consideration of the applied dose alone.

Studies of methylhexaneamine in supplements and geranium oil.

A number of supplements are now available which are sold as fat burners or pre-workout boosters and contain stimulants which are banned in sport. Many contain methylhexaneamine under one of many pseudonyms including Geranamine, geranium oil or extract, or a number of chemical names such as 1,3-dimethylpentylamine. This has resulted in many athletes returning an adverse finding and having sanctions imposed. Other stimulants such as caffeine, phenpromethamine, synefrine, and phenethylamines are also to be found in supplements. This communication shows that geranium oils do not contain methylhexaneamine and that products labelled as containing geranium oil but which contain methylhexaneamine can only arise from the addition of synthetic material. Since the usual dose of methylhexaneamine is large, the drug is excreted at relatively high amounts for more than 29 h, the time for which the excretion was studied.

Elicitation of the immune response to p-phenylenediamine in allergic patients: the role of dose and exposure time.

BACKGROUND: Usage of hair dye products containing p-phenylenediamine (PPD) is a concern for PPD-allergic individuals. OBJECTIVES: The present study investigates the role of dose and exposure time on elicitation of allergic contact dermatitis under conditions of permanent hair dyeing. METHODS: Elicitation responses after application of a typical hair dye product containing 2% PPD for 30 min followed by rinsing were analysed in 38 PPD-allergic individuals with a documented history of hair dye-related allergy. Skin binding experiments in vitro were performed to distinguish the dose available for elicitation from the dose applied. RESULTS: A positive reaction was elicited in 20 of 20 patients with grades ++ to +++ and 12 of 18 with grade + according to the classification of the International Contact Dermatitis Research Group. Under conditions of diagnostic patch testing (48 h exposure), the dose available for elicitation is more than 10-fold higher compared with the dose available for hair dyeing (30-min exposure, rinsing of product). CONCLUSIONS: This investigation demonstrates that under simulated hair dye use conditions the actual exposure to PPD is more than an order of magnitude lower than under diagnostic patch testing, although sufficient to elicit a clearly noticeable reaction in 84% of PPD patch test-positive individuals.

Skin metabolism of aminophenols: human keratinocytes as a suitable in vitro model to qualitatively predict the dermal transformation of 4-amino-2-hydroxytoluene in vivo.

4-Amino-2-hydroxytolune (AHT) is an aromatic amine ingredient in oxidative hair colouring products. As skin contact occurs during hair dyeing, characterisation of dermal metabolism is important for the safety assessment of this chemical class. We have compared the metabolism of AHT in the human keratinocyte cell line HaCaT with that observed ex-vivo in human skin and in vivo (topical application versus oral (p.o.) and intravenous (i.v.) route). Three major metabolites of AHT were excreted, i.e. N-acetyl-AHT, AHT-sulfate and AHT-glucuronide. When 12.5 mg/kg AHT was applied topically, the relative amounts of each metabolite were altered such that N-acetyl-AHT product was the major metabolite (66% of the dose in comparison with 37% and 32% of the same applied dose after i.v. and p.o. administration, respectively). N-acetylated products were the only metabolites detected in HaCaT cells and ex-vivo whole human skin discs for AHT and p-aminophenol (PAP), an aromatic amine known to undergo N-acetylation in vivo. Since N-acetyltransferase 1 (NAT1) is the responsible enzyme, kinetics of AHT was further compared to the standard NAT1 substrate p-aminobenzoic acid (PABA) in the HaCaT model revealing similar values for K(m) and V(max). In conclusion NAT1 dependent dermal N-acetylation of AHT represents a 'first-pass' metabolism effect in the skin prior to entering the systemic circulation. Since the HaCaT cell model represents a suitable in vitro assay for addressing the qualitative contribution of the skin to the metabolism of topically-applied aromatic amines it may contribute to a reduction in animal testing.

Warm and cold parental reproductive environments affect seed properties, fitness, and cold responsiveness in Arabidopsis thaliana progenies.

Conditions in the parental environment during reproduction can affect the performance of the progenies. The goals of this study were to investigate whether warm or cold temperatures in the parental environment during flowering and seed development affect Arabidopsis thaliana seed properties, growth performance, reproduction and stress tolerance of the progenies, and to find candidate genes for progeny-related differences in stress responsiveness. Parental plants were raised at 20 degrees C and maintained from bolting to seed maturity at warm (25 degrees C) or cold (15 degrees C) temperatures. Analysis of seed properties revealed significant increases in nitrogen in seeds from warm temperature and significant increases in lipids and in the ratio of alpha-linolenic to oleic acid in seeds from the cold parental environment. Progenies of the warm parental environment showed faster germination rates, faster root elongation growth, higher leaf biomass and increased seed production at various temperatures compared with those from the cold parental environment. This indicates that under stable environmental conditions, progenies from warm parental environments had a clear adaptive advantage over those from cold parental environments. This parental effect was presumably transmitted by the higher nitrogen content of the seeds developed in warm conditions. When offspring from parents grown at different temperatures were exposed to chilling or freezing stress, photosynthetic yield recovered faster in progenies originating from cold parental environments. Cold acclimation involved up-regulation of transcripts of flavanone 3-hydroxylase (F3H) and pseudo response regulator 9 (PRR9) and down-regulation of growth-associated transcription factors (TFs) NAP and AP2domain containing RAP2.3. NAP, a regulator of senescence, and PRR9, a temperature-sensitive modulator of the circadian clock, were probably involved in mediating parent-of-origin effects, because they showed progeny-related expression differences under chilling. Because low temperatures also delay senescence, cold responsiveness of NAP suggests that this factor is linked with the regulatory network that is important for environmental acclimation of plants.

Donepezil for Alzheimer's disease in clinical practice--The DONALD Study. A multicenter 24-week clinical trial in Germany.

This multicenter open-label clinical trial was designed to investigate the safety and efficacy of donepezil, a selective acetylcholinesterase inhibitor, in the treatment of Alzheimer's disease (AD) in routine clinical practice in Germany. A total of 237 patients with mild-to-moderate AD were treated with donepezil for 24 weeks, 186 completed the study according to the protocol. In the completer group, mean MMSE score for efficacy showed an improvement from baseline of +1.6 points at week 12 (95% CI +1.1 to +2.1) and of +1.1 points at week 24 (95% CI +0.5 to +1.7). In more than 80% of the patients, global tolerability was rated to be very good or good. There were only insignificant effects on ECG parameters. This study confirms the results obtained in previous double-blind trials, which showed that donepezil is effective and well tolerated in patients with mild-to-moderately severe AD.

Serum IFN-gamma and IL-10 levels are associated with disease progression in non-obese diabetic mice.

BACKGROUND: The goal of the present study was to determine whether cytokines in the peripheral blood of naive NOD mice correlate with the disease process and thereby would provide a marker for monitoring disease activity. METHODS: Female NOD mice (5, 10 and 14-16 weeks of age) were investigated in a cross-sectional study. In the group of 14-16-week-old mice, non-diabetic and diabetic mice were analysed as different subgroups. The Th1 cytokine (IFN-gamma) and the Th2 cytokine (IL-10) were quantified in serum by sandwich enzyme-linked immunosorbent assay (ELISA). Pancreatic mRNA for IFN-gamma and IL-10 was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) from the same animals. RESULTS: Serum levels of IFN-gamma were initially low but increased with age in NOD mice, reaching the highest levels at diabetes onset (p<0.002 compared to 10 weeks). A similar rise was noted in IFN-gamma gene expression in pancreatic lesions. In contrast, an early peak of serum IL-10 levels was observed in non-diabetic NOD mice (10 weeks) at a stage where non-destructive insulitis occurs. With increasing age a continuous loss of IL-10 until progression towards diabetes was observed. The pancreatic IL-10 mRNA expression correlated with serum IL-10 changes. As a consequence, the ratio of IFN-gamma/IL-10, reflecting the Th1/Th2 balance in the serum, was significantly increased in diabetic compared to non-diabetic NOD mice (p<0.005). CONCLUSION: These results demonstrate, for the first time, that an increased Th2 pattern in the non-diabetic stage preceding a Th1 shift is associated with the development of diabetes in naive NOD mice. Serum cytokines correlate with disease progression and pancreatic cytokine expression during prediabetes. Soluble cytokines measured in the periphery are therefore promising surrogate markers of diabetes development.

[Cycle disorders, polydipsia, decline of vision. Is it the fault of the hypophysis?]. / Zyklusstörungen, Polydipsie, Visusminderung. Ist die Hypophyse schuld?

Apart from partial or complete insufficiency, further diseases of the pituitary with clinical impact are those associated with hormone overproduction such as acromegaly, Cushing's disease and prolactinoma. The cardinal symptom of pituitary insufficiency or disruption of the female cycle, loss of libido or sexual potency reflecting a lesion of the gonadotrophic axis. Hyperprolactinemia also results in a loss of gonadotrophic function. Macroadenomas of the pituitary gland often give rise to visual field defects, so that an ophthalmological work-up is a must. With the exception of hyperprolactinemia, measurement of basic hormone levels is not always sufficient to detect pituitary diseases. For the establishment of hormone deficiency, therefore, stimulation tests, and for detection of hypersecretion suppression tests, are carried out. Owing to the increasing utilization of cranial MRI, incidental detection of intrasellar tumors, the so-called incidentalomas, is becoming ever more common, and these lesions require at least an endocrinological investigation.

High-performance liquid chromatographic determination of bradykinin in saliva: a critical review and a new method.

Because of difficulties or dubious results with previously published methodologies, a new semi-automated HPLC method with UV absorbance detection was developed and applied to the determination of bradykinin (BK) in human saliva. The new method consisted of an uncomplicated sample preparation involving the addition to saliva of an equal volume of 0.1 M orthophosphoric acid to stabilize BK, vortex-mixing, centrifugation, and separation, followed by chromatography of the supernatant phase on a C8, 150x3.9-mm (I.D.) stainless steel column. The mobile phase was composed of 19% acetonitrile/0.1% trifluoroacetic acid at flow-rate of 0.4 ml/min. Using UV detection at 220 nm, the detection limit was 1 ng/ml for the BK standard, and 7 ng/ml for the assay of endogenous salivary BK. The orthophosphoric acid initially added to the saliva allowed BK to be stabilized from enzymic degradation at 20 degrees C for 5 days and at 4 degrees C for 60 days. Assignment made to the peak with the chromatographic properties of salivary BK was confirmed by HPLC-MS with an electrospray interface. This paper presents a new method that is reproducible, reliable and allows kinetic studies of salivary BK to be performed for clinical investigations.

T cells ignore aniline, a prohapten, but respond to its reactive metabolites generated by phagocytes: possible implications for the pathogenesis of toxic oil syndrome.

The most basic arylamine, aniline, belongs to a class of compounds notorious for inducing allergic and autoimmune reactions. In 1981 in Spain, many people succumbed to toxic oil syndrome (TOS), a disease caused by ingestion of cooking oil contaminated with aniline. Indirect evidence points toward an immune pathogenesis of TOS driven by T lymphocytes, but it is unclear to which antigens these cells could react. Here, using the popliteal lymph node (PLN) assay in mice, we analyzed the sensitizing potential of aniline, its metabolites, and some of the aniline-coupled lipids detected in the contaminated cooking oil. Whereas aniline itself and its non-protein-reactive metabolites nitrobenzene, p-aminophenol and N-acetyl-p-aminophenol, failed to elicit PLN responses, its reactive metabolites nitrosobenzene and N-hydroxylaniline did. The aniline-coupled lipids, namely, linoleic anilide and linolenic anilide, and a mixture of fatty acid esters of 3-(N-phenylamino)-1,2-propanediol, all implicated in TOS, induced significant PLN responses, whereas the respective aniline-free lipids, linoleic acid, linolenic acid, and triolein, did not. Hence, the aniline moiety plays a crucial role in the immunogenicity of the aniline-coupled lipids of TOS. PLN responses to the reactive aniline metabolites and the one aniline-coupled lipid that was tested, linolenic anilide, were T-cell-dependent. Secondary PLN responses to nitrosobenzene were detectable not only after priming with nitrosobenzene but, in some experiments, also after priming with linolenic anilide. This suggests that the aniline moiety was cleaved from the aniline-coupled lipid and metabolized to the intermediate nitrosobenzene that generated the prospective neoantigens. Consistent with this, in lymphocyte proliferation tests in vitro, T cells primed to nitrosobenzene reacted in anamnestic fashion to white bone marrow cells (WBMCs) pulsed with aniline. Hence, we propose that aniline is a prohapten that can be metabolized by WBMCs, which form neoantigens that are recognized by T cells. The possible significance of these findings for the pathogenesis of TOS is discussed.

The effect of sudden failure of a rotary blood pump on left ventricular performance in normal and failing hearts.

We investigated the hemodynamic effect of regurgitation (or back-flow) due to sudden failure of a rotary blood pump (diagonal pump). Seven healthy sheep (Group C) and 7 with chronic heart failure (Group F) were studied. Chronic heart failure was obtained by intracoronary injection of microspheres several weeks earlier. Left ventricular function and ventricular efficacy were assessed by the pressure-volume relationship. The back-flow through the stopped pump was significantly lower in Group F (2.3 +/- 0.34 L/min) than in Group C (2.8 +/- 0.33 L/min). Mean aortic blood pressure dropped significantly from 68.3 +/- 9.65 to 61.9 +/- 9.75 mm Hg in Group C and from 62.5 +/- 9.12 to 51.5 +/- 9.08 in Group F but remained stable during the 15 min period of pump stop. Parameters of left ventricular contractility (preload recruitable stroke work) dropped significantly in both groups, remained stable during the pump stop, and returned to baseline values 30 min after the end of back-flow. The ventricular efficacy (in terms of energy transfer) was tolerant against this acute volume overload even in the failing hearts. Sudden pump failure of a rotary blood pump leads to an acute depression of the hemodynamic state and myocardial contractility. However, this depression remained stable over 15 min, did not lead to further deterioration of the animals, and was completely reversible.

Growth factor-induced phosphoinositide 3-OH kinase/Akt phosphorylation in smooth muscle cells: induction of cell proliferation and inhibition of cell death.

OBJECTIVE: The signaling pathways mediating proliferation and apoptosis in vascular smooth muscle cells (VSMC) are not well established. It has previously been shown that activation of the phosphoinositide 3-OH kinase (PI3K)/Akt pathway or the ERK 1/2 pathway can mediate anti-apoptotic function in different cell types. This study determined the specific contribution of the PI3K/Akt and ERK pathway in the regulation of apoptosis and proliferation of VSMC. METHODS AND RESULTS: Incubation of rat VSMC with FCS, insulin or IGF-1 time-dependently stimulated the phosphorylation of Akt, however FCS but not insulin or IGF-1 activated the MAP-kinase ERK 1/2. Moreover, insulin inhibited H(2)O(2)-induced apoptosis via the Akt pathway as demonstrated by pharmacological inhibition of the PI3K or overexpression of a dominant negative Akt mutant. In contrast, FCS inhibited H(2)O(2)-induced apoptosis via the Akt and also the ERK pathway. FCS, but not insulin or IGF-1 induced VSMC proliferation, suggesting that Akt activation is necessary but not sufficient for VSMC proliferation. FCS-induced proliferation of VSMC was only mediated via the Akt pathway and not the ERK pathway. CONCLUSIONS: These results define a link between cell proliferation and programmed cell death in VSMC via the same signal transduction pathway, namely activation of the serine/threonine kinase Akt, which may have significant implication for the development of vascular diseases or remodeling.

Orally administered lead chloride induces bias of mucosal immunity.

The hypothesis that lead disturbs gut immune functions upon oral ingestion was tested. Long-term exposure to oral PbCl(2)for 10 days caused persistent downregulation of TGF-beta mRNA levels in intestinal tissue. PbCl(2) also disturbed oral tolerance induction to the dietary antigen ovalbumin. Upon challenge with an immunizing dose of ovalbumin and rechallenge of draining lymph node cells in vitro, tolerance induction was partially suppressed in animals exposed to oral PbCl(2). This was shown by increased proliferation to antigenic stimulus, increased production of IFN-gamma and decreased secretion of TGF-beta. In conclusion, we show for the first time that oral exposure to PbCl(2)has a significant effect on the gut immune system, demonstrated by a bias of the cytokine pattern towards Th(1)and by disturbed oral tolerance mechanisms.

Determination of defensin HNP-1, HNP-2, and HNP-3 in human saliva by using LC/MS.

The mass spectral profiling of saliva by liquid chromatography mass spectrometry in relation to particular types of pain is being examined. The aim is to develop a profile that could be useful for the assessment of patients and their treatment programs, as well as identifying unknown compounds observed in saliva. Defensin human neutrophil peptide-1 (HNP-1) and defensin HNP-2 were identified and confirmed, whereas defensin HNP-3 was tentatively identified. Linear calibration range of defensin HNP-1 and HNP-2 was 0.25 to 3 microg/ml with R(2) values of > 0.99 for both. The detection limit for defensin HNP-1 and HNP-2 was estimated at 0.1 microg/ml. The healthy subjects surveyed in this study had readily measurable salivary concentrations of defensin HNP-1 (8.6 +/- SD 8.0 microg/ml) and defensin HNP-2 (5.6 +/- SD 5.2 microg/ml).