Kende Lőrincz

In vivo second-harmonic generation and ex vivo coherent anti-stokes raman scattering microscopy to study the effect of obesity to fibroblast cell function using an Yb-fiber laser-based CARS extension unit

Nonlinear microscopy techniques are being increasingly used to perform in vivo studies in dermatology. These methods enable us to investigate the morphology and monitor the physiological process in the skin by the use of femtosecond lasers operating in the red, near‐infrared spectral range (680–1,300 nm). In this work we used two different techniques that require no labeling: second harmonic generation (SHG) for collagen detection and coherent anti‐Stokes Raman scattering (CARS) to assess lipid distribution in genetically obese murine skin. Obesity is one of the most serious public health problems due to its high and increasing prevalence and the associated risk of type 2 diabetes and cardiovascular diseases. Other than these diseases, nearly half of patients with diabetes mellitus suffer from dermatological complications such as delayed wound healing, foot ulcers and several other skin changes. In our experiment we investigated and followed the effects of obesity on dermal collagen alterations and adipocyte enlargement using a technique not reported in the literature so far. Our results indicate that the in vivo SHG and ex vivo CARS imaging technique might be an important tool for diagnosis of diabetes‐related skin disorders in the near future. Microsc. Res. Tech. 78:823–830, 2015.

Diet-induced obesity skin changes monitored by in vivo SHG and ex vivo CARS microscopy

Obesity related metabolic syndrome and type 2 diabetes have severe consequences on our skin. Latest developments in nonlinear microscopy allow the use of noninvasive, label free imaging methods, such as second harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS), for early diagnosis of metabolic syndrome-related skin complications by 3D imaging of the skin and the connective tissue. Our aim was to study effects of various types of diet-induced obesity in mice using these methods. We examined mice on different diets for 32 weeks. The collagen morphology was evaluated four times in vivo by SHG microscopy, and adipocytes were examined once at the end of experiment by ex vivo CARS method. A strong correlation was found between the body weight and the adipocyte size, while we found that the SHG intensity of dermal collagen reduces considerably with increasing body weight. Obese mice on high-fat diet showed worse results than those on high-fat - high-fructose diet. Animals on high-fructose diet did not gain more weight than those on ordinary diet despite of the increased calorie intake, but their collagen damage was nonetheless significant. Obesity and high sugar intake damages the skin, mainly the dermal connective tissue and subcutaneous adipose tissue, which efficiently can be monitored by in vivo SHG and ex vivo CARS microscopy.

Development of lupus erythematosus during infliximab therapy

Infliximab is a TNFα inhibiting recombinant monoclonal antibody, which provides an efficient therapeutic opportunity in the treatment of psoriasis and other immune-mediated inflammatory diseases. It is well tolerated and improves quality of life significantly. The authors present a case of drug-induced lupus erythematosus as a possible side effect of this medication. The patient developed psoriasis 9 years ago when she was on beta-receptor blocker therapy. The symptoms deteriorated despite topical and systemic treatments and, therefore, biological therapy was introduced. In the third year of treatment drug-induced lupus erythematosus was diagnosed on the background of general symptoms. After cessation of the biologic treatment a low dose corticosteroid therapy was introduced which proved to be effective. Symptoms as well as pathological laboratory parameters showed an improvement. The authors conclude that biologicals are effective and safe in the treatment of psoriasis, nevertheless, they have risks too. To reduce side effects a meticulous follow-up of patients is essential. Any general symptom requires careful examination since they might be linked to serious side effects of the biological therapy.

Development of lupus erythematosus during infliximab treatment

Infliximab is a TNFα inhibiting recombinant monoclonal antibody, which provides an efficient therapeutic opportunity in the treatment of psoriasis and other immune-mediated inflammatory diseases. It is well tolerated and improves quality of life significantly. The authors present a case of drug-induced lupus erythematosus as a possible side effect of this medication. The patient developed psoriasis 9 years ago when she was on beta-receptor blocker therapy. The symptoms deteriorated despite topical and systemic treatments and, therefore, biological therapy was introduced. In the third year of treatment drug-induced lupus erythematosus was diagnosed on the background of general symptoms. After cessation of the biologic treatment a low dose corticosteroid therapy was introduced which proved to be effective. Symptoms as well as pathological laboratory parameters showed an improvement. The authors conclude that biologicals are effective and safe in the treatment of psoriasis, nevertheless, they have risks too. To reduce side effects a meticulous follow-up of patients is essential. Any general symptom requires careful examination since they might be linked to serious side effects of the biological therapy.

Quantitative Analysis on Ex Vivo Nonlinear Microscopy Images of Basal Cell Carcinoma Samples in Comparison to Healthy Skin

Basal cell carcinoma (BCC) is the most frequent malignant neoplasm in the Caucasian population. There are several therapeutic options for BCC, but surgical excision is considered gold standard treatment. As BCCs often have poorly defined borders, the clinical assessment of the tumor margins can be challenging. Therefore, there is an increasing demand for efficient in vivo imaging techniques for the evaluation of tumor borders prior to and during surgeries. In the near future, nonlinear microscopy techniques might meet this demand. We measured the two-photon excitation fluorescence (TPEF) signal of nicotinamide adenine dinucleotide hydride (NADH) and elastin and second harmonic generation (SHG) signal of collagen on 10 ex vivo healthy control and BCC skin samples and compared the images by different quantitative image analysis methods. These included integrated optical density (IOD) measurements on TPEF and SHG images and application of fast Fourier transform (FFT), CT-FIRE and CurveAlign algorithms on SHG images to evaluate the collagen structure. In the BCC samples, we found significantly lower IOD of both the TPEF and SHG signals and higher collagen orientation index utilizing FFT. CT-FIRE algorithm revealed increased collagen fiber length and decreased fiber angle while CurveAlign detected higher fiber alignment of collagen fibers in BCC. These results are in line with previous findings which describe pronounced changes in the collagen structure of BCC. In the future, these novel image analysis methods could be integrated in handheld nonlinear microscope systems, for sensitive and specific identification of BCC.

Confirmation of the role of a KRT5 mutation and successful management of skin lesions in a patient with Galli-Galli disease

Galli–Galli disease (GGD) is a rare autosomal dominant genodermatosis, which is considered an acantholytic variant of Dowling–Degos disease (DDD). It is characterized by reticular, lentigo-like hyperpigmentation on the groins, axillae and large folds, occurring between puberty and early adulthood. Histology shows increased pigmentation of the basal layer, acanthosis with moderate to severe acantholysis, and finger-like rete ridges that result in antler-like patterns of epidermal downgrowths. Despite the presence of acantholysis, skin fragility has not been observed. There is no curative treatment for GGD, but symptoms may be managed by topical corticosteroids, topical and systemic retinoids, intense pulsed light or ablative laser therapy. The aforementioned clinical presentation corresponds to the DDD1 (OMIM 179850) variant caused by KRT5 mutations. Several mutations have been identified in the first exon of KRT5, all of which lead to premature termination codons (PTC), both in sporadic and familial cases. Most cases were reported from Germany, in which c.418dupA was described as the most common mutation in patients with acantholytic DDD. Further mutations were described in Spanish, Arabic, Asian-American, Chinese and Indian patients. In some cases, acantholysis was not observed, and it has also been reported that acantholysis may not necessarily be present at all times or at all anatomical sites. In many cases, both from Europe and Asia, an association between DDD/ GGD and a KRT5 mutation could not be detected. Recently, novel mutations in the POGLUT1 and POFUT1 genes of the Notch signalling pathway have also been identified as causative of DDD in German and Chinese familial and sporadic cases. A 74-year-old Hungarian man presented with a 30year history of a slowly progressing skin condition. Physical examination revealed poikiloderma and widespread pigmented lesions on the patient’s neck and trunk, and on the flexor surfaces of the extremities with small hypopigmented papules, and hyperpigmented macules, which occasionally became inflamed, forming psoriasiform papules over the reticular pattern (Fig. 1a). The patient’s father, grandfather and two brothers had the same ‘ocelot-like’ skin. Histopathology revealed increased pigmentation of the basal layer, finger-like rete ridges in the suprapapillary epidermis, and presence of acantholysis (Fig. 1c,d). A diagnosis of GGD was suggested. Both the inflamed skin condition and pigmentation improved significantly after the introduction of acitretin 25 mg daily (Fig. 1b), which was later reduced to 25 mg alternate daily. When the therapy was temporarily discontinued, the lesions suddenly worsened. Following ethics approval and informed consent, genetic analysis was performed. A heterozygous duplication of the base 418 of KRT5 was found, which causes a frameshift mutation resulting in a PTC in codon 179 (c.418dupA; p.Ile140AsnfsX39; Fig. 1e). This is the first Hungarian case of GGD with a clear genetic background. The KRT5 mutation c.418dupA (p.Ile140AsnfsX39) has been previously identified by Hanneken et al. as being the most common mutation Correspondence: Dr Norbert M. Wikonk al, Department of Dermatology, Venereology and Dermato-oncology, Semmelweis University, 41 M aria Street, 1085 Budapest, Hungary E-mail: wikonkal.norbert@med.semmelweis-univ.hu

Ex vivo nonlinear microscopy imaging of Ehlers–Danlos syndrome-affected skin

Ehlers–Danlos syndrome (EDS) is the name for a heterogenous group of rare genetic connective tissue disorders with an overall incidence of 1 in 5000. The histological characteristics of EDS have been previously described in detail in the late 1970s and early 1980s. Since that time, the classification of EDS has undergone significant changes, yet the description of the histological features of collagen morphology in different EDS subtypes has endured the test of time. Nonlinear microscopy techniques can be utilized for non-invasive in vivo label-free imaging of the skin. Among these techniques, two-photon absorption fluorescence (TPF) microscopy can visualize endogenous fluorophores, such as elastin, while the morphology of collagen fibers can be assessed by second-harmonic generation (SHG) microscopy. In our present work, we performed TPF and SHG microscopy imaging on ex vivo skin samples of one patient with classical EDS and two patients with vascular EDS and two healthy controls. We detected irregular, loosely dispersed collagen fibers in a non-parallel arrangement in the dermis of the EDS patients, while as expected, there was no noticeable impairment in the elastin content. Based on further studies on a larger number of patients, in vivo nonlinear microscopic imaging could be utilized for the assessment of the skin status of EDS patients in the future.

Photosensitivity of murine skin greatly depends on the genetic background: clinically relevant dose as a new measure to replace minimal erythema dose in mouse studies.

Artificial UV irradiation of murine skin is a frequently used method for testing photosensitivity, study carcinogenesis and photoprotective effects of different compounds. However, doses of UV radiation and mouse strains used in experiments vary greatly. The genetic background of mice may influence the photosensitivity as melanin content, pigmentation and hair cycle parameters are dissimilar. Doses of UV are often expressed in relation to the minimal erythema dose (MED) that was not necessarily determined for the given strain. We set out to standardize the method of measuring photosensitivity in three commonly used mouse strains, C57BL/6N, Balb/c and SKH‐1. We found that MED may not be determined for some strains as erythema development in mice with diverse genotypes differs greatly. We measured the oedema response in vivo and ex vivo by using OCT. Given the strain‐specific variability of erythema, we introduced Clinically Relevant Dose (CRD) as a new term to replace MED in experiments, to describe the lowest dose that triggers a perceptible skin reaction in mice. Not only the CRD but the proportion of erythema and oedema were different in strains examined. C57BL/6N mice display skin reactions at the lowest UVB dose, while SKH‐1 hairless mice show changes, mostly oedema, after higher doses of UVB. The cellular composition and skin thickness were examined by histopathology. IL‐1beta and IL‐6 levels in skin correlated with the increasing doses of UVB. Despite the variations in the degree of erythema and oedema, no major differences in cytokine expressions were seen among various strains of mice.

Analysis of skin aging during long term PUVA treatment on mice

A PUVA fényterápiás kezelést számos bôrgyógyászati kórképben alkalmazzák sikeresen évtizedek óta. A fényterápia rövid és hosszú távú kockázatait tekintve korántsem egységesek az irodalmi adatok. Narrow-band UVB fényforrásnál számtalan állatokon végzett kísérlet igazolta az UV sugárzás okozta bôrrák képzôdés és bôröregedés kockázatának növekedését. Ezzel szemben a PUVA terápia ilyen jellegû hatásairól kevés adat áll rendelkezésre kísérletes körülmények között végzett vizsgálatokból. Epidemiológiai jellegû vizsgálatok eredményei azonban felhívják a figyelmet az elôbbi káros hatások kialakulására hosszú távú PUVA kezelést követôen. A szerzôk krónikus PUVA kezelés hatásainak vizsgálatát és szemléltetését tûzték ki célként egérmodell alkalmazásával.