Introduction

Psoriasis is a chronic, immune-mediated inflammatory skin disease characterized by longer or shorter periods of relapses and remissions. The prevalence of psoriasis is high in Western Europe (1.07–3.46%), but in Central Europe is higher (0.62–5.32%) [1, 2]. The illness can occur at any age, but most patients present with the condition before 35 years old. The disease has different appearance and shape (plaque, nail, scalp, guttate, inverse, pustular, erythrodermic psoriasis), and in rare cases, it can affect the joints (psoriatic arthritis) [3, 4].

The skin manifestations in patients with psoriasis are characterized by hyperproliferation of keratinocytes with abnormal differentiation and infiltration of inflammatory cells, mainly activated T cells in the epidermis and papillary part of the dermis. These symptoms are frequently categorized from mild, through moderate to severe type (Fig. 1). The classification is depending on the clinical severity of the lesions, the percentage of affected body surface area, and patient quality of life [5,6,7].

Fig. 1
figure 1

(Source: Dr. M. Moezzi)

Asymptomatic (A), mild–moderate (B), and severe (C) psoriatic skin lesions on the patients’ trunk

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The diagnosis of psoriasis is usually clinical based on dermatological examination, and in rarely cases, a skin biopsy is needed to establish the differential diagnosis. Commonly used tools for classification of psoriasis include the Psoriasis Area and Severity Index (PASI), body surface area (BSA), and the Dermatology Life Quality Index, with a score of more than 10 on each of these parameters indicating moderate-to-severe psoriasis. In case of joint involvement, radiological examinations (X-ray, ultrasound, MRI) should be necessary to confirm diagnosis. Currently, specific blood tests and/or radiographic findings often not available that reliably confirmed the diagnosis or give an opinion on the course of the disease [9, 10].

More and more is known about the pathogenesis of the process, and in parallel, new and better treatment options are now already available for patients. According to recent research proinflammatory cyto- and chemokines (C3, C4, IL-23, TNFα, IFNα, IL2,6,8,12,15,17,22,23), adhesion and growth factors (Cx26 and 30, EGF, HSP27, and 60, TGFβ), different T cells (CD4 + , CD8 + , DCs) and their receptors usually act in that inflammatory and proliferative processes which will result the well-known psoriatic skin signs and symptoms [11,12,13]. Considering psoriasis is a chronic, recurrent disease, it often requires lifelong treatment. The choice of treatment is largely influenced by the severity of the disease, the presence of other co-morbidities, and the patient's access to health care. In accordance with agreed protocols, mild-to-moderate psoriasis can be treated with local agents (topical therapy with corticosteroids, vitamin D analogues, retinoids, and phototherapies). In some moderate cases and in every severe case, systemic treatment (drug administration in monotherapy or in combination) should be required [6, 12, 13].

Regarding that only few molecular targets are yet known to regulate the pathogenesis of the disease, controlling of it is not an easy task. One reason for this is that, unfortunately, the key pathogenetic mechanisms are still unknown. Therefore, the known mechanisms do not always apply to all individuals. At least 15–20% of patients do not respond to the latest targeted antipsoriatic agents, so in such cases or in exacerbations, a return to conventional, conservative steroid drug therapy is necessary [12, 13]. Biological response modifiers are highly effective in psoriasis, and according to their action mechanism, two main classes target T cells or cytokines. In addition, second-generation retinoids are well-used drugs and have relatively few local side effects [6, 12, 13].

Differential scanning calorimetry (DSC) is a now widely used thermoanalytical method that measures changes in the physical or chemical properties of biological samples as a function of temperature. In DSC, the principle of measurement is to compare the heat flow rate of a sample and an inert substance heated or cooled at the same rate. Changes in the sample are associated with changes in the differential thermal heat flow, which is then recorded as a peak value. The first comparative measurements with blood samples from patients were carried out by Garbett and his team in the early 2000s [14, 15]. This research group was the first to suggest that DSC measurements could show disease and condition-specific variations and it could have significance in clinical diagnosis. After our animal research experiments, in the beginning of the 2000-years fruitful collaborations between our research group and clinicians from different professions have led to a very broad spectrum of medical applications of plasma thermoanalysis. Thus, it has been successfully applied in patients with various solid tumorous diseases or other systemic inflammatory diseases, such as pancreatitis or psoriasis [16,17,18,19,20,21,22,23]. Moreover, according to the PASI scoring system different severity of psoriasis have been thermoanalytically detectable (denaturation temperature, calorimetric enthalpy) in our previous studies [10, 16].

The aim of the present study was to investigate thermal changes of blood plasma by DSC in patients who are asymptomatic or have moderate or severe symptoms following cytostatic, retinoid, or biological response modifier therapy.

Materials and methods

Patient selection

Psoriatic patients (equal shares of both sexes, aged from 21 to 75 years) with a mean age of 50.15 ± 5.5 were selected in the study. As controls, 10 age and sex-matched healthy volunteers’ samples (mean age 48.6 ± 3.2 years) were measured. To define the severity of symptoms, three groups were established based on PASI (Psoriasis Area Severity Index) scoring system as previously described [12]. As brief, PASI 0 meant symptomless, while PASI 1–15 showed mild–moderate symptoms, and severe symptoms were observed if PASI was over 15. According to administration of antipsoriatic monotherapy, the patients were divided into three groups: cytostatic therapy, retinoid treatment, and biological response modifiers receiving persons. The groups were established according to the response to treatment: symptomless after drugs, mild-moderated symptoms after medications, and severe symptoms despite drug therapies (n = 10 in each group). The type, the dosage, and timing of drug treatment were done in accordance with the current dermatological protocols. The study protocol was approved by regional ethical committee of Pécs University (4077/2011).

Blood sample collection and preparation

Peripheral blood samples were collected from each patient (n = 30) and from healthy controls (n = 10). Blood samples were collected into the Vacutainer tubes containing EDTA (1.5 mg mL−1 of blood) centrifuged at 1.600 g for 15 min at 4 °C to separate plasma fraction from cell components. Native plasmas were stored at − 80 °C until DSC measurement.

Calorimetric measurement

The thermal unfolding of the human plasma components was monitored by SETARAM Micro DSC-II calorimeter as previously described [2, 12]. As brief, all experiments were conducted between 0 and 100 °C. The heating rate was 0.3 K min−1 in all cases. Conventional Hastelloy batch vessels were used during the denaturation experiments with 850 μL sample volume in average. Reference sample was normal saline (0.9% NaCl). The sample and reference samples were equilibrated with a precision of ± 0.1 mg. The repeated scan of denatured sample was used as baseline reference, which was subtracted from the original DSC curve. We have plotted the heat flow (DSC-II is a heat flux instrument with hermetically closed vessels) in the function of temperature. Calorimetric enthalpy was calculated from the area under the) heat flow curve by using two-point setting SETARAM peak integration.

Statistical analysis

All results are given in mean values ± standard error of the mean (SEM). Data were analyzed with one-way ANOVA. The level of significance was set at p < 0.05.

Results

This study investigated the thermal changes of human blood plasma following different systemic drug treatment on patients suffering in psoriasis. The macroscopic appearance of psoriasis skin symptoms can be seen on patients in Fig. 1.

In Fig. 2, the healthy human blood plasma DSC scan is observable, where the heat flow reaches its maximum at the characteristic transition temperature (Tm) of albumin around 63 °C. The half width of the heat flow is small, indicating the more compact structure of this protein in intact case. Meanwhile, the blood plasma’s calorimetric enthalpy was in the range which is usual in case of this kind of biological materials (ΔH ~ 1.29 ± 0.07 Jg−1).

Fig. 2
figure 2

Healthy human blood plasma DSC scan (control, endotherm deflection downward)

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In case of psoriatic patients who undergone therapeutic treatment, we distinguished three groups (see Table 1). In the denaturation scans in case of those who becoming asymptomatic after systematic treatment (see Fig. 3), the biological response modification treatment was the closest to the control in shape and Tm. The cytostatic treatment increased the Tm of albumin by ~ 2 °C (63 → 65), but the Tm of retinoid treatment was between the previous two transitions with a definite shoulder around 71 °C which is the contribution of globulin (see Fig. 3) and had an endotherm at 85 °C assigned to transferrin [30]. In psoriatic patients without symptoms, the calorimetric enthalpy was increased after cytostatic or retinoid treatments compared to control blood plasma samples (1.50 ± 0.03 Jg−1 vs. 1.29 ± 0.07 Jg−1). In contrast, the biological response modification treatment caused the smallest changes in this thermal parameter (1.49 ± 0.03 Jg−1 vs. 1.29 ± 0.07 Jg−1, see Table 1).

Table 1 The thermal denaturation parameters of blood plasma in patients with psoriasis following different systemic drug monotherapy. (Tm/°C peak temperature of denaturation („melting” point), ΔH/Jg−1 calorimetric enthalpy of transitions normalized for total sample mass, data are in mean ± s.d.)
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Fig. 3
figure 3

Average DSC thermal curves in psoriatic patients who becoming asymptomatic after systemic cytostatic, retinoid or biological agent therapies (endotherm deflection downward)

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The denaturation scans of psoriatic patients with mild–moderate symptoms can be seen in Fig. 4. The run, shape, and Tm in case of biological drug treatment were also like to the control. The Tm increased in retinoid as well as cytostatic treatment, indicating a stronger albumin structure. In case of cytostatic drug intervention, the shoulder in the DSC scan remained nearly at the same temperature as it was in patients without symptoms after drug treatment (~ 74 °C, see Figs. 3 and 4). Comparing these patient groups, the calorimetric enthalpy after the biological response modification treatment showed the closest value to the healthy plasma controls (1.45 Jg−1 ± 0.02 vs. 1.29 ± 0.07 Jg−1) while cytostatic monotherapy caused the greatest changes in the calorimetric enthalpy (1.60 ± 0.08 Jg−1 vs. 1.29 ± 0.07 Jg−1, Table 1).

Fig. 4
figure 4

Thermal changes in psoriatic patients' blood plasma who has mild–moderate symptoms following systemic cytostatic, retinoid, or biological drug treatments (endotherm deflection downward)

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The psoriatic patients with severe symptoms revealed the most interesting effects (see Fig. 5.). Surprisingly, the Tm in case of biological response modification treatment increased significantly, compared to the control, and reached the highest value during the whole procedure. The retinoid answer remained the nearly same as in all classification. The Tm decreased in cytostatic treatment and the shoulder disappeared from the vicinity of 71 °C. From these groups, the calorimetric enthalpy of this transition was the highest after cytostatic treatment, while the nearest to the control value following the biological response modification therapy (see Table 1).

Fig. 5
figure 5

DSC scans of psoriatic patients' blood plasma who has severe symptoms despite systemic cytostatic, retinoid or biological drug therapies (endotherm deflection downward)

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Discussion

Psoriasis is a genetically strongly determined, T-cell-associated inflammatory disease. Based on recent studies, over 40 regions of the genome and more than 400 single-nucleotide polymorphisms associated with psoriasis have been identified [24]. In fact, it is multifactorial disease that, in addition to involve hereditary elements, is caused by environmental factors (trauma, infection, drugs, stress, smoking and alcohol consumption), dysfunction of endocrine system, and an increased immune response of the skin. The chronic disease is mainly associated by proinflammatory T-helper1 cells and cytokine-producing T-helper17 cells which in healthiest are modulated by regulatory T cells [2].

Diagnosis is based on the clinically used PASI score by a dermatologist, which is essentially a qualitative estimate, not a validated, quantified diagnostic criterion [6]. From 2011, our research group started to investigate new possible diagnostic measures for patients with psoriasis. These examinations detected firstly the human blood plasma thermal changes by DSC technique in untreated and following different therapies on patients with psoriasis. This first study concluded that cytostatic and retinoid agents are essential and useful in the treatment of psoriasis, but that a new generation of therapy is represented by a specific monoclonal drug formulation effective in severe or therapy-resistant cases. Structural changes in blood plasma measured by DSC showed that biological response modifiers caused an improvement in plasma thermal stability for all measurable thermodynamic parameters [6, 31]. Our results are considered novel because we firstly reported such calorimetric changes in psoriatic patients’ blood plasma who have different symptoms despite antipsoriatic monotherapy or who become asymptomatic following these treatments. However, similar observations have been made by our other researcher group in a clinical study where patients suffered with pancreatitis were examined as in other systematic inflammatory disease [25].

As a further step, next to the well-known general properties of blood plasma, the aim of the present study was to use DSC to detect the effect of psoriasis drugs on patients' symptoms. In the current study, it was also observable that the significant decrease in the melting temperatures is a sign of less thermal stability of the macromolecular structure. Examining patients who became asymptomatic with treatment and patients who received the biological response modification treatment was the closest to the control in shape and Tm, indicating that this treatment gave the most specific results. Moreover, similar to the healthy plasma cases denaturation (around 76 °C) is assigned to C3 protein, IgA and IgG fragments, and albumin. In the treated cases, the same compounds in the 72–80 °C temperature range (the biological moderate state was put here because of its high enthalpy contribution) could be identify. However, the scan with the cytostatic treatment shows a broad transition at about 80 °C. Following retinoid and biological therapy, the calorimetric enthalpy slightly fluctuates, but in cytostatic treated an extra enthalpy jump has been observed, which involves the contributions of IgG and transferrin.

In contrast, in psoriasis patients with severe symptoms even with different treatments, Tm was surprisingly significantly increased in the biological response modifier treatment compared to the control, reaching the highest value in the whole procedure. Meanwhile, severe cases showed less shift in plasma curves after systemic cytostatic treatment. The pronounced heat capacity change between denatured and native states is also an indicator of the structural change in disease compared with the healthy controls.

These results are considered novel; no results have been reported so far describing such changes in blood plasma in patients with psoriasis consistent with symptoms associated with antipsoriatic treatment. However, the present observations are also in line with our previous results in which we have reported similar findings in chronic disease (e.g., pancreatitis) as in other systemic inflammatory diseases [2, 12]. More and more research groups have been investigated blood plasma changes in patients yet who treated for cancer, inflammatory disease or other causes [26,27,28,29]. But this study is a continuation of our earlier psoriasis patient’ study showing that there is a detectable difference in blood plasma for symptoms that change after treatment. There is no doubt that the importance and role of DSC analysis in understanding the stability of biological systems in the human body (such as blood plasma) is indisputable, and further studies are needed in the future.