Barrier lipid composition and response to plasma lipids: A direct comparison of mouse dorsal back and ear skin

The skin of the ear and the back are frequently selected sites in skin research using mouse models. However, distinct responses to treatment have been described between these two sites in several studies. Despite the crucial role of the stratum corneum (SC) in the skin barrier function of both dorsal back and ear skin, it remains unclear whether differences in lipid composition might underlie altered responses. Here, we compared the skin morphology and the barrier lipid composition of the ear with the back skin of wild-type mice. The ear contained more corneocyte layers in the SC and its barrier lipid composition was enriched with sphingosine ceramide subclasses, especially the short ones with a total chain length of 33-34 carbons. The free fatty acid (FFA) profile in the ear skin shifted towards shorter chains, significantly reducing the mean chain length to 23.3 vs 24.7 carbons in the back skin. In line, FFA species in the ear displayed a twofold increase in unsaturation index (P < .001). Gene expression in the ear skin revealed low expression of genes involved in lipid synthesis and uptake, indicating a reduced metabolic activity. Finally, the effects of hypercholesterolaemia on SC FFA composition was compared in ear and back skin of apolipoprotein E knockout (APOE^−/−) mice. Interestingly, the FFA profile in APOE^−/− ear skin was minimally affected, while the FFA composition in the back skin was markedly changed in response to hypercholesterolaemia. In conclusion, ear and back skin have distinct barrier lipids and respond differently to elevated plasma cholesterol.     

SKIN LESIONS IN HIV-POSITIVE AND HIV-NEGATIVE PATIENTS IN SOUTH INDIA

Background. Various dermatologic conditions have been reported to occur with increased frequency in human immunodeficiency virus (HIV)-positive individuals, but there are only a few studies comparing the prevalences of skin diseases in HIV-positive patients with those in matched HIV-negative controls. Methods. Skin lesions in 129 HIV-positive patients and 258 HIV-negative controls were studied prospectively over an 18-month period from October 1991 to March 1993. Results. Oral Candida, tinea cruris, and ichthyosis were significantly more common in HIV-positive patients compared to controls. Several other dermatologic conditions were found only in the HIV-positive group. Conclusions. The pattern of skin lesions in Indian patients with HIV infection may be different from that in the West.     

Human papillomavirus-associated induction of human β-defensins in anal intraepithelial neoplasia

Background  Antimicrobial peptides and proteins (AMPs) are widely distributed effector molecules of the innate immune system with well-known antibacterial activity. However, there is a paucity of information regarding antiviral effects of AMPs.
Objectives  The present study was performed to analyse expression of AMPs in human papillomavirus (HPV)-associated anal skin lesions of human immunodeficiency virus (HIV)-positive men who have sex with men (MSM), a special high-risk group for persistent HPV infections and anal dysplasia.
Methods  Skin lesions were analysed for the presence of LL-37, RNase 7, and human β-defensin (hBD)-1, hBD-2 and hBD-3. Moreover, HPV typing and HPV DNA load determination for HPV types 6, 11, 16, 18, 31 and 33 were performed to evaluate possible correlations between expression of AMPs and lesional HPV types.
Results  Skin biopsies of 45 HIV-positive MSM with anal intraepithelial neoplasia (AIN), anal condylomata acuminata or unaffected anal mucosa, as well as condylomata acuminata of eight HIV-negative MSM, were analysed for AMP mRNA expression. Additionally, immunohistochemical analysis for hBD-2 and hBD-3 was performed in a total of 45 samples. hBD-2 and hBD-3 gene and protein expression was significantly increased in both AIN and condyloma, whereas LL-37, RNase 7 and hBD-1 gene expression did not differ significantly from unaffected anal mucosa. AMP expression correlated neither with the number of HPV types nor with the high-risk and low-risk HPV DNA loads of the quantified types. No significant differences in AMP expression were observed in condylomata of HIV-positive and HIV-negative MSM.
Conclusions  hBD-2 and hBD-3 expression was shown to be significantly upregulated in HPV-associated anal skin lesions of both HIV-positive and HIV-negative MSM. Their biological significance in the innate immunity against these lesions needs further research.     

Untersuchungen über die Zusammensetzung des palmaren Hautoberflächenfettes im Altersablauf und deren biochemische Grundlagen

Zusammenfassung Es wurde die Zusammensetzung des palmaren Hautoberflächenfettes im Altersablauf untersucht. Die Ergebnisse haben gezeigt, daß der Gehalt an freien Fettsäuren (FFS) bedeutend höher als derjenige der Triglyceride ist. Beide stehen in inverser Relation zueinander. Die FFS werden durch hydrolytischen Abbau der Triglyceride gebildet. Der Abbau wird vornehmlich durch Bakterienlipasen bewirkt. Die Werte für die in Wachs- und Cholesterinestern gebundenen Fettsäuren (FS) sind mit Ausnahme der 31–40 jährigen relativ konstant. Die Menge an freiem Cholesterin ist bei der höchsten Altersgruppe am größten. Squalen konnte nur in Spuren nachgewiesen werden. Über das Verhältnis von gesättigten und ungesättigten FS wird berichtet. Die am häufigsten und mengenmäßig am stärksten vorkommende Komponente in allen Lipidklassen ist die Palmitinsäure. Die biochemischen Grundlagen und die mögliche biologische Funktion der Hautoberflächenlipide werden diskutiert.

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Investigations of the composition of palmar skin surface fat in relation to age and its biochemical fundamentals

Summary The composition of the palmar skin surface lipids in relation to age was investigated. The results have shown that the content of free fatty acids (FFA) is much higher than that of triglycerides. Both are in inverse relation to each other. FFA are formed by hydrolytic breakdown of the triglycerides. Breakdown of the triglycerides is provided by bacterial lipases. The values of the esterified fatty acids (FA) in wax- and sterol esters are relatively constant with exception of the 31–40 age group. The amount of free cholesterol is highest in the oldest age group. Squalen was only in small amounts present. The proportion of saturated and unsaturated fatty acids is reported. The main component in all lipid classes was palmitic acid. The biochemical fundamentals and the possible biological function of the skin surface lipids are discussed.

     

The importance of free fatty acid chain length for the skin barrier function in atopic eczema patients

An important feature of atopic eczema (AE) is a decreased skin barrier function. The stratum corneum (SC) lipids – comprised of ceramides (CERs), free fatty acids (FFAs) and cholesterol – fulfil a predominant role in the skin barrier function. In this clinical study, the carbon chain length distribution of SC lipids (FFAs and CERs) and their importance for the lipid organization and skin barrier function were examined in AE patients and compared with control subjects. A reduction in FFA chain length and an increase in unsaturated FFAs are observed in non‐lesional and lesional SC of AE patients. The reduction in FFA chain length associates with a reduced CER chain length, suggesting a common synthetic pathway. The lipid chain length reduction correlates with a less dense lipid organization and a decreased skin barrier function. All changes are more pronounced in lesional SC compared with non‐lesional skin. No association was observed between lipid properties and filaggrin mutations, an important predisposing factor for developing AE. The results of this study demonstrate an altered SC lipid composition and signify the importance of these changes (specifically regarding the CER and FFA chain lengths) for the impaired skin barrier function in AE. This provides insights into epidermal lipid metabolism as well as new opportunities for skin barrier repair.     

Analyses of skin surface lipid in patients with microbially associated skin disease

The composition of the total skin surface lipid, free fatty acids and fatty acid of triglyceride was used in discriminant analyses to classify patients with severe acne vulgaris (AY), pityriasis versicolor (PV), seborrhoeic dermatitis (SD) and atopic dermatitis (AD). It was not possible to discriminate between the PV and SD patients but alt the lipid groups gave a satisfactory separation between AV, PV + SD and AD patients. A similar separation was obtained when the sex of the patient was studied but not when both sex and disease were taken into account. The results suggest that there are fundamental differences in the composition of skin lipid between patients with microbially associated diseases, but the precise role of the organisms has not been ascertained.     

人免疫缺陷病毒阳性人群人乳头状瘤病毒感染的型别分析

目的 了解人免疫缺陷病毒（HIV）阳性人群人乳头状瘤病毒（HPV）感染状况、型别分布特征及与HIV阴性人群的差异。 方法 选择门诊患有尖锐湿疣或主诉近期与可疑尖锐湿疣患者有性接触者作为研究对象,包括HIV阳性组62例和HIV阴性组2 716例,采用基因芯片技术对外生殖器部位皮肤黏膜样本进行HPV检测,采用SPSS 19.0统计软件进行χ2检验。 结果 HIV阳性组中,HPV总感染率74.19%（46/62）,高危型HPV感染率67.74%（42/62）,低危型HPV感染率58.06%（36/62）;HIV阴性组三者感染率分别为42.30%、29.57%、24.71%,两组比较,差异有统计学意义（均P < 0.01）。HIV阳性组男性HPV检出率高达92.11%（35/38）,和HIV阴性组男性检出率相比（37.38%,382/1 022）差异有统计学意义（χ2 = 45.98,P < 0.01）。低危型HPV在HIV阳性组检出率前3位为HPV6（37.10%,23/62）、43和11型,在HPV阴性组检出率排序相同但HPV6的检出率仅11.12%（302/2 716）。高危型HPV在HIV阳性组检出率前5位依次是HPV16（22.58%,14/62）、52、66、58和18型,在HIV阴性组前5位依次为HPV16（7.77%,211/2 716）、58、56、66和52型。HPV亚型重叠感染情况,HIV阳性组最多八重,HIV阴性组最多九重,三重及以上感染者,HIV阳性组65.21%（30/46）,HIV阴性组16.71%（192/1 149）,差异有统计学意义。 结论 HIV阳性人群HPV总感染率、高危型HPV感染率、低危型HPV感染率、男性HPV感染率以及HPV多重感染率均明显高出HIV阴性组,值得临床高度重视。     

Surfactants have multi-fold effects on skin barrier function

Background

The stratum corneum (SC) is responsible for the barrier properties of the skin and the role of intercorneocyte skin lipids, particularly their structural organization, in controlling SC permeability is acknowledged. Upon contacting the skin, surfactants interact with the SC components leading to barrier damage.

Objective

To improve knowledge of the effect of several classes of surfactant on skin barrier function at three different levels.

Methods

The influence of treatments of human skin explants with six non-ionic and four ionic surfactant solutions on the physicochemical properties of skin was investigated. Skin surface wettability and polarity were assessed through contact angle measurements. Infrared spectroscopy allowed monitoring the SC lipid organization. The lipid extraction potency of surfactants was evaluated thanks to HPLC-ELSD assays.

Results

One anionic and one cationic surfactant increased the skin polarity by removing the sebaceous and epidermal lipids and by disturbing the organization of the lipid matrix. Another cationic surfactant displayed a detergency effect without disturbing the skin barrier. Several non-ionic surfactants disturbed the lipid matrix organization and modified the skin wettability without any extraction of the skin lipids. Finally two non-ionic surfactants did not show any effect on the investigated parameters or on the skin barrier.

Conclusions

The polarity, the organization of the lipid matrix and the lipid composition of the skin allowed describing finely how surfactants can interact with the skin and disturb the skin barrier function.

     

The effect of testosterone and anabolic steroids on the skin surface lipids and the population of Propionibacteria acnes in young postpubertal men

The effect of testosterone and anabolic steroids on skin surface lipids and the population of Propionibacteria acnes (P. acnes) was studied in power athletes. The subjects used self-administered high doses of testosterone and anabolic steroids during a 12-week strength training period. After 8 weeks' use of hormones the amount of dissolved skin surface lipids (SSL), and the Colony Forming Units/cm2 (CFU/cm2) of P. acnes had increased (p less than 0.01). The percentage values of dissolved SSL constituents changed. The cholesterol (CHO) and also the relative values of free fatty acids (FFA) increased. SSL constituents obtained by collection on absorbent paper likewise changed the dissolved constituents. It was concluded that high doses of testosterone and anabolic steroids may increase the SSL, the P. acnes population, and the percentage of the CHO and FFA of the skin surface lipids in healthy young men.     

Molecular interactions of plant oil components with stratum corneum lipids correlate with clinical measures of skin barrier function

Plant‐derived oils consisting of triglycerides and small amounts of free fatty acids (FFAs) are commonly used in skincare regimens. FFAs are known to disrupt skin barrier function. The objective of this study was to mechanistically study the effects of FFAs, triglycerides and their mixtures on skin barrier function. The effects of oleic acid (OA), glyceryl trioleate (GT) and OA/GT mixtures on skin barrier were assessed in vivo through measurement of transepidermal water loss (TEWL) and fluorescein dye penetration before and after a single application. OA's effects on stratum corneum (SC) lipid order in vivo were measured with infrared spectroscopy through application of perdeuterated OA (OA‐d₃₄). Studies of the interaction of OA and GT with skin lipids included imaging the distribution of OA‐d₃₄ and GT ex vivo with IR microspectroscopy and thermodynamic analysis of mixtures in aqueous monolayers. The oil mixtures increased both TEWL and fluorescein penetration 24 h after a single application in an OA dose‐dependent manner, with the highest increase from treatment with pure OA. OA‐d₃₄ penetrated into skin and disordered SC lipids. Furthermore, the ex vivo IR imaging studies showed that OA‐d₃₄ permeated to the dermal/epidermal junction while GT remained in the SC. The monolayer experiments showed preferential interspecies interactions between OA and SC lipids, while the mixing between GT and SC lipids was not thermodynamically preferred. The FFA component of plant oils may disrupt skin barrier function. The affinity between plant oil components and SC lipids likely determines the extent of their penetration and clinically measurable effects on skin barrier functions.     

The lipid alterations in the stratum corneum of dogs with atopic dermatitis are alleviated by topical application of a sphingolipid-containing emulsion

Background. Atopic dermatitis (AD) results from an altered skin barrier associated with defects in the lipid composition of the skin. Dogs with AD present similar clinical symptoms to humans, and may be a useful model for investigations into AD. Aim. To analyse the changes occurring in the lipids of the stratum corneum (SC) of dogs with AE after 3 weeks of topical treatment with an emulsion containing ceramides, free fatty acids (FFAs) and cholesterol (skin lipid complex; SLC). Methods. Nonlesional SC was collected by tape stripping from control and treated areas. Free and protein‐bound lipids were purified, and the various classes were isolated by column chromatography, analysed by thin‐layer chromatography and assayed. Results. Ceramides, FFA and cholesterol were all found to be lower in the skin of untreated dogs with AD than in normal dogs, and the topical treatment resulted in significantly increased values for ceramides. Conversely, only trace amounts of glucosylceramides were present in normal SC, but a high concentration (27 μg per mg protein) was detected in canine atopic SC, which disappeared after treatment with SLC. There was a heterogeneous distribution of all of the lipids in the different layers of canine atopic SC, which was more pronounced for protein‐bound than for free lipids. Following topical treatment, the protein‐bound lipid content normalized. Conclusions. Topical treatment with SLC resulted in a significant improvement of the lipid biosynthesis of keratinocytes in atopic dogs, thereby potentially enabling the formation of a tighter epidermal barrier.     

Some effects of fats with dietary carbohydrates on the lipids on the surface of the skin

Young men and women were given diets containing various carbohydrate mixtures given with either sunflower seed oil or cream and the triglyceride and cholesterol concentration on the surface of the skin above the right scapula was determined. In men, the triglyceride level on the surface of the skin was reduced by sunflower seed oil. In young women the nature of the dietary constituents seemed to have no marked effect on the lipids on the surface of the skin.     

Early-stage HIV infection and hepatitis C virus infection are associated with elevated serum porphyrin levels

Background: Porphyria cutanea tarda is known to be associated with HIV infection and hepatitis C virus (HCV). Objective: Our purpose was to evaluate whether early infection with HIV, with or without HCV infection, is associated with elevated serum porphyrin levels. Methods: Serum porphyrin levels were measured in samples obtained from 103 patients with early HIV infection. The results were compared with those of 89 late-stage HIV–positive patients and 78 HIV-negative patients. Results: The highest median porphyrin level was in early-stage HIV-positive/HCV-positive samples, followed in decreasing order by those in early-stage HIV-positive/HCV-negative, late-stage HIV-positive/HCV-positive, late-stage HIV-positive/HCV-negative, HIV-negative/HCV-positive, and HIV-negative/HCV-negative groups. Elevated porphyrin levels were independently associated with early-stage HIV infection (P < .0001) and HCV infection (P = .03). Conclusion: This finding suggests abnormal porphyrin metabolism is most noticeable in early-stage HIV infection; it becomes less severe with the progression of HIV disease. (J Am Acad Dermatol 1998;39:956-9.)     

Topically applied fatty acids are elongated before incorporation in the stratum corneum lipid matrix in compromised skin

In several skin diseases, both the lipid composition and organization in the stratum corneum (SC) are altered which contributes to the impaired skin barrier function in patients. One of the approaches for skin barrier repair is treatment with topical formulations to normalize SC lipid composition and organization. Vernix caseosa (VC), a white cheesy cream on the skin during gestational delivery, has shown to enhance skin barrier repair. In this study, we examined how a fatty acid (FA) containing formulation mimicking the lipid composition of VC interacts with the lipid matrix in the SC. The formulation was applied on ex vivo human skin after SC removal. Subsequently, the ex vivo human skin generated SC during culture. The effect of FA containing formulations on the lipid organization and composition in the regenerated SC was analysed by Fourier transform infrared (FTIR) spectroscopy and liquid chromatography mass spectroscopy (LC/MS), respectively. FTIR results demonstrate that the FAs are intercalated in the lipid matrix of the regenerated SC and partition in the same lattice with the endogenous SC lipids, thereby enhancing the fraction of lipids forming an orthorhombic (very dense) packing in the SC. LC/MS data show that the topically applied FAs are elongated before intercalation in the lipid matrix and are thus involved in the lipid biosynthesis in the skin.     

Free fatty acids in the early inflammatory papule of acne vulgaris

The role of free fatty acids (FFA) in the pathogenesis of acne is disputed. Several studies have failed to show an increase in FFA concentration in skin surface lipids (SSL) from acne patients. It has been suggested that a marked increase in FFA concentration in a single sebaceous follicle may lead to the inflammatory lesion in acne, and because SSL samples are usually taken from a large area, this localized increase would be swamped by the lipid from the surrounding normal follicles. We therefore used a micro-cylinder method to obtain SSL from early acne papules and we compared this with SSL from nearby ‘normal’ skin in 19 patients. The samples were analysed by quantitative thin layer chromatography. We found no significant difference in the mean FFA concentration in SSL obtained from acne papules and normal skin. Our findings do not support the idea that the inflammation of acne is due to an increase in FFA concentration in skin lipids.     

Prevalence, Incidence, and Risk Factors for Human Papillomavirus 16 Seropositivity in Australian Homosexual Men

BACKGROUND:: Human papillomavirus 16 (HPV16) has been causally associated with approximately 70% of anal cancers. This cancer is markedly increasing among homosexual men. There is limited knowledge of the epidemiology and natural history of anal HPV infection in homosexual men. METHODS:: Behavioral data and sera for antibodies to HPV16 L1 were collected annually for 1427 HIV-negative and 245 HIV-positive Australian homosexual men. Seroprevalence, seroincidence, and risk factors were calculated. RESULTS:: Among HIV-negative men, 25.4% were HPV16 seropositive at baseline compared with 44.3% of HIV-positive men. HPV16 seroincidence was 3.1/100 person-years among HIV-negative men and 1.3/100 person-years among HIV-positive men. Seroincidence among HIV-negative men remained >3% per year until 45 years of age, before declining. In multivariate analyses of data from HIV-negative men, seroprevalent HPV16 was associated with sexual risk behaviors and seropositivity for several viral sexually transmissible infections. Seroincident HPV16 was associated with younger age and unprotected anal intercourse with HIV-positive partners. Among men who predominantly practiced insertive anal intercourse, circumcision was associated with a 57% reduction in seroincident HPV16 (hazard ratio = 0.43, 95% confidence interval: 0.21-0.88, P = 0.021). CONCLUSIONS:: HPV16 seroincidence remained common in men until their mid 40s suggesting that vaccination may be protective in sexually active young gay men. Both HPV16 seroprevalence and seroincidence correlated well with markers of higher risk sexual activity, particularly receptive anal sexual practices. An association between circumcision and decreased HPV16 seroconversion in HIV-negative men who preferred the insertive position in anal sex was observed.     

Comparison of structure and organization of cutaneous lipids in a reconstructed skin model and human skin: spectroscopic imaging and chromatographic profiling

The use of animals for scientific research is increasingly restricted by legislation, increasing the demand for human skin models. These constructs present comparable bulk lipid content to human skin. However, their permeability is significantly higher, limiting their applicability as models of barrier function, although the molecular origins of this reduced barrier function remain unclear. This study analyses the stratum corneum (SC) of one such commercially available reconstructed skin model (RSM) compared with human SC by spectroscopic imaging and chromatographic profiling. Total lipid composition was compared by chromatographic analysis (HPLC). Raman spectroscopy was used to evaluate the conformational order, lateral packing and distribution of lipids in the surface and skin/RSM sections. Although HPLC indicates that all SC lipid classes are present, significant differences are observed in ceramide profiles. Raman imaging demonstrated that the RSM lipids are distributed in a non‐continuous matrix, providing a better understanding of the limited barrier function.     

Lipid profile in actinic keratosis and basal cell carcinoma

BACKGROUND: The lipid content of the skin and its changes are important in the pathogenesis of many disorders affecting the skin, particularly actinic keratosis (AK) and basal cell carcinoma (BCC). METHODS: Cholesterol, phospholipid, triglyceride, and total lipid levels were studied in paired lesional (AK and BCC) and nonlesional intact skin of 13 patients with AK and 12 patients with BCC. Serum concentrations of the same lipid fractions studied in the skin were investigated in AK and BCC patients and in 11 healthy, age-matched controls. RESULTS: Levels of all lipid fractions were increased in both AK and BCC skin. When AK and BCC skin were compared with each other, a significant increase in phospholipids (p < 0.02) and total lipids (p < 0.01) was found in BCC. Serum cholesterol (p < 0.001), phospholipid (p < 0.001), triglyceride (p < 0.05), and total lipid (p < 0.001) concentrations of AK patients were significantly higher than those of the control group. When BCC and controls were compared, a significant increase in phospholipids and total lipids (p < 0.001) was seen. Serum cholesterol in BCC patients was significantly lower (p < 0.001) and serum phospholipid levels were significantly higher (p < 0.05) than those in the AK group. CONCLUSIONS: An increase in the metabolically active serum phospholipid fraction is reflected in elevated neoplastic tissue phospholipid. This produces altered proportions between lipid fractions in tumorous areas and may result in changes in the intact nature of the cellular membrane, spread, and malignant proliferation.     

Mucocutaneous manifestations in Japanese HIV-positive hemophiliacs

BACKGROUND: Although various mucocutaneous manifestations have been reported in patients infected with HIV by sexual transmission or intravenous drug use, the prevalence and characteristics of skin disorders in HIV-positive hemophiliacs coinfected with hepatitis C virus (HCV) have rarely been described. OBJECTIVE: The purpose of this study was to clarify the characteristics of skin disorders in HIV-positive hemophiliacs and to identify differences in comparison with other HIV-positive groups. METHODS: A prospective study of the prevalence of mucocutaneous manifestations in 110 Japanese hemophiliacs (53 HIV-positive hemophiliacs including 24 AIDS and 57 HIV-negative hemophiliacs) was performed from July 1997 to July 1998. RESULT: None of the hemophiliacs developed Kaposi's sarcoma or sexually transmitted skin diseases. Eosinophilic folliculitis was observed in 3 AIDS patients. The incidence of folliculitis, common warts, seborrheic dermatitis, generalized eczema, oral candidiasis and herpes zoster was higher in HIV-positive than in HIV-negative hemophiliacs (p < 0.05). Although anti-HCV antibody was positive in all HIV-positive hemophiliacs, HCV-related dermatoses such as lichen planus and porphyria cutanea tarda were not observed. CONCLUSION: Although Kaposi's sarcoma and sexually transmitted skin diseases such as molluscum contagiosum, condyloma, and scabies are frequently associated with HIV, they were not found in the HIV-positive hemophiliacs in our study. HIV infection-related mucocutaneous manifestations are influenced not only by the presence of HIV but also by other factors such as the mode of transmission and sexual habit.     

Pityrosporum ovale and seborrhoeic dermatitis in HIV-seropositive and HIV-seronegative men. Quantitative cultures and serological studies

Background Previous studies of quantitative determinations of Pityrosporum ovale in HIV-seropositive patients with and without seborrhoeic dermatitis are contradictory. Objective To investigate the number of P. ovale among HIV-seronegalive and HIV-seropositive men using contact plates containing cow's milk as the main lipid source and to analyse sera with ELISA for IgG antibodies to a P. ovale protein extract. Results There was no difference in the number of P. ovale between healthy controls and HIV-positive patients with and without seborrhoeic dermatitis. HIV-seropositive patients had significantly lower IgG antibody litres to a P. ovale protein extract compared with healthy individuals.