Skin Lesion Samples Research Articles

Genetic diagnosis of multiple affected tissues in a patient with McCune–Albright syndrome

McCune-Albright syndrome (MAS) is a sporadic disorder characterized by the classic triad of polyostotic fibrous dysplasia, café-au-lait' skin pigmentation, and hyperfunctional endocrinopathy. It is caused by embryonic somatic mutations leading to the substitution of His or Cys for Arg at amino acid 201 of the alpha-subunit of the signal transduction protein Gs (Gsalpha). A 32-year-old man was diagnosed as McCune-Albright syndrome with the following findings: polyostotic fibrous dysplasia, café-au-lait' spots and acromegaly. An ultrasonic examination showed that he had left-pleural effusion, which disappeared after almost a year without special treatment. Genomic DNA was isolated from the peripheral blood, bone tissue, skin lesion and pleura samples of the patient. Then PCR and direct sequencing were performed. An activating mutation of the Gsalpha gene (Arg201Cys) was found in the genomic DNA isolated from the peripheral blood and the bone tissue, but not in genomic DNA isolated from the skin and pleura samples.

Early increase in TNF-alpha-containing mast cells in skin lesions

We have evaluated the numbers and immunohistochemical positivity for tumor necrosis factor (TNF) alpha of the mast cells in (a) 40 skin samples collected at autopsy from subjects who had survived for a few seconds to 1 h, (b) 10 samples of post-mortem skin lesions and (c) 10 surgical biopsies of healthy skin. Sections were treated with fluoresceinated avidin, to tag mast cell granules, followed by indirect immunohistochemistry for TNF-alpha with polyclonal primary and rhodaminated secondary antibodies. We could confirm a progressive increase in mast cell numbers, which became significant 1 h after trauma. More important, the fraction of mast cells positive for TNF-alpha increased progressively in the same time period and became significantly higher than controls in specimens collected more than 15 min after trauma. Samples from post-mortem lesions had significantly fewer mast cells and fewer TNF-alpha-positive cells than any other group of samples. The results suggest that mast cells are quickly recruited to an injured site in response to trauma and upregulate their TNF-alpha content, which can play an early role in directing tissue response to injury. The forensic pathologist can take advantage from this behavior of mast cells for the evaluation of the timing of early vital lesions.

AXENIC PROMASTIGOTE FORMS OF LEISHMANIA (VIANNIA) LAINSONI AS AN ALTERNATIVE SOURCE FOR LEISHMANIA ANTIGEN PRODUCTION

The present study demonstrates that axenic cultures of Leishmania (Viannia) lainsoni produce larger cell masses in NNN-LIT medium, as well as higher amounts of total proteins in cell extracts, than Leishmania (Leishmania) amazonensis. Antigenicity of L. (V.) lainsoni whole promastigotes is similar to that of L. (L.) amazonensis, as demonstrated by an indirect immunofluorescence diagnostic test using sera from human patients and dogs infected with visceral leishmaniasis. Infectivity of the L. (V.) lainsoni strain used in the present work was demonstrated by the detection by transmission-electron microscopy of tissue amastigotes in skin lesion samples from an experimentally infected hamster. Incubation of lesion fragments in NNN-LIT medium allowed us to obtain promastigote forms, which could be cultivated successfully in vitro. lsoenzyme analysis of such promastigotes confirmed the parasite strain as L. (V.) lainsoni, as compared to other Leishmania reference strains. Our data indicate that L. (V.) lainsoni is a useful alternative source for antigen production as well for use in assays that depend on large cell volumes of Leishmania spp. parasites.

Proapoptotic and antiapoptotic markers in cutaneous T-cell lymphoma skin infiltrates and lymphomatoid papulosis.

In cutaneous T-cell lymphoma (CTCL) lesions, both reactive T cells and malignant T cells intermingle. The disease progression is mostly slow. Recent evidence suggests that even if clinical remission is reached, malignant cells persist and a relapse follows sooner or later. To wha extent tumour cell apoptosis occurs in the skin lesions either due to the reactive T cells or t therapeutic efforts is not known. To determine the extent of tumour cell apoptosis and the expression of proapoptotic an antiapoptotic markers in serial skin lesion samples from patients with CTCL, and to compare th findings with those in patients with lymphomatoid papulosis (LyP). Thirty-four skin samples were obtained from 12 patients with CTCL at the time o diagnosis and at a mean of 1.6, 3 and 6 years later. The patients received psoralen plus ultraviolet (PUVA), electron beam or cytostatic treatments. In addition, fresh post-treatment samples fro three patients with CTCL undergoing PUVA therapy were obtained. For comparison, skin biopsies o five patients with LyP were studied. Immunohistochemical demonstration of the expression of th following markers was performed on formalin-fixed skin sections: Fas (CD95), Fas ligand (FasL) bcl-2, granzyme B, the tumour-suppressor protein PTEN and the effector caspase, caspase-3. Th malignant cells were identified morphologically, and apoptotic cells were identified with th terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling method on parallel sections. In untreated CTCL lesions, apoptotic lymphocytes were extremely rare, and no increase in the number of apoptotic cells was observed after any of the treatments used. In LyP, apoptotic cell were more frequent, comprising on average 5% of the infiltrate. The apoptosis-associated marker Fas, FasL, caspase-3 and granzyme B were expressed by morphologically neoplastic cells in CTCL and by large atypical cells in LyP, with no significant differences. However, only a few reactive cell in CTCL infiltrates expressed granzyme B while about 10% of the corresponding cells were positive in LyP. The expression of antiapoptotic bcl-2 was more frequent in CTCL than in LyP, while PTE expression was high in both instances. The number of bcl-2 + cells tended to decrease after therapy When comparing the findings between the first and the last samples, a decrease in the number of bcl-2+ cells and an increase in Fas+ cells was associated with disease progression, despite therapy, while the opposite was true for remissions. Apoptosis was found to be a rare event in CTCL lesions irrespective of precedin therapy During patient follow-up, no significant differences in the expression of apoptotic marker was observed while in most cases a lower level of antiapoptotic bcl-2 expression was observed after all types of therapies and in association with disease progression when compared with high expression in the untreated lesions. The absence of apoptosis and high expression of bcl-2 together with a low expression of apoptosis-inducing granzyme B in the reactive lymphocytes in CTC could explain the chronic nature of the disease and the poor response to therapy, while th more frequent occurrence of granzyme B and apoptosis together with a lower level of expressio of bcl-2 by the large atypical cells in LyP could contribute to the favourable outcome of the latter.

Infections cutanées graves à Streptococcus pyogenes

Streptococcus pyogenes is the first bacteria encountered in severe cutaneous infections in children. They enclose erysipela (papillar derma concerned more than hypoderma, lymphatic involvment) and necrotising fasciitis NF (focus on fascia and muscles with extension to hypoderma and reticular derma; venous thrombosis; hypodermic and aponevrotic necrosis). A skin lesion is the entrance of infection: varicella lesions are a major factor of NF. In France, sporadic cases in children are observed. The annual incidence of S. pyogenes invasive diseases is 1/100 000 children under 5 years of age and 0.6/100 000 children under 15 years of age. In North America, resurgence has been notified during the past ten years with mortality and morbidity due to NF and toxic shock syndromes. Interaction between bacteria and host, natural reservoir, explains the physiopathology. During the past ten years, some serotypes have become more invasive and virulent. Any cutaneous lesion interrupt the dermal barrier. Bacterial wall, proteins M and adhesins permit colonisation. Four pyrogenic exotoxins are superantigens; some facilitate inflammation, tissular lesions and shock; other participate to bacterial extension. In young children, immune response is immature. Albeit causal link between non-steroids anti-inflammatory drugs and NF in varicella children was not clearly demonstrated, caution should be kept in mind. Diagnosis of erysipela is clinical: sudden appearance of an inflammatory zone, most often on legs, with high fever and pain; sometime peripheral surelevation, lymphangitis, adenopathia. Other aspects open discussion with NF. In NF are in favour, added to high fever, huge pain, erythema and oedema: rapid extension of lesions, cutaneous hypoesthesia appearance, gap between intense severe general status (toxic shock syndrome) and paucity of local signs. NF is a medico-surgical emergency. Early surgery with complete excision of necrotic tissues permit survival. Magnetic resonance is useful in subacute NF. Microbiological diagnosis is possible in 20 to 80% of cases, using combined methods. Blood cultures identify the bacteria in 5 to 20%, skin lesion samples in 30%. Local samples are less useful even with modern techniques. Therapeutic strategy depends on initial diagnosis. Intra-venous antibiotics are necessary: penicillin (G, A or M) is first line therapy. In erysipela, ten days allow a rapid cure without sequellae. In NF, antibiotics are associated with intensive care and surgery. A late diagnosis, a too late surgery explain 16 to 36% of deaths encountered.

Lesions of so-called flank biting and necrotic ear syndrome in pigs.

Skin lesions were established on the flanks of pigs aged 10 to 12 weeks, in the areas of the ribs, the mid-upper leg, the shoulder and the tuber ischii. Skin lesions on the ear tips, known as porcine necrotic ear syndrome, were detected in weaners throughout the raising period. By the histological examination of 50 samples of skin lesions from the flank, two stages of epidermal and dermal changes were established; the milder stage had epidermal changes with marked acanthosis, rete ridges formed deep in the dermis and mild infiltrates of mononuclear cells in the layers around the capillaries in the dermis; the more severe stage had ulcers, under which there were infiltrates of polymorphonuclear cells. The same histological changes were observed in 23 samples of skin lesions taken from the tips of ears with necrotic ear syndrome. On healthy skin from the flank and ear tip, micrococci were the dominant organism, whereas on skin lesions from both the affected areas staphylococci were dominant. Since the histological lesions on the flanks were identical to those in necrotic ear syndrome, which has been associated with Staphylococcus hyicus infection, the pathogenesis of the lesions on the flanks is probably the same.

Arsenic Enhancement of Skin Neoplasia by Chronic Stimulation of Growth Factors

Although numerous epidemiological studies have shown that inorganic arsenicals cause skin cancers and hyperkeratoses in humans, there are currently no established mechanisms for their action or animal models. Previous studies in our laboratory using primary human keratinocyte cultures demonstrated that micromolar concentrations of inorganic arsenite increased cell proliferation via the production of keratinocyte-derived growth factors. As recent reports demonstrate that overexpression of keratinocyte-derived growth factors, such as transforming growth factor (TGF)-alpha, promote the formation of skin tumors, we hypothesized that similar events may be responsible for those associated with arsenic skin diseases. Thus, the influence of arsenic in humans with arsenic skin disease and on mouse skin tumor development in transgenic mice was studied. After low-dose application of tetradecanoyl phorbol acetate (TPA), a marked increase in the number of skin papillomas occurred in Tg.AC mice, which carry the v-Ha-ras oncogene, that received arsenic in the drinking water as compared with control drinking water, whereas no papillomas developed in arsenic-treated transgenic mice that did not receive TPA or arsenic/TPA-treated wild-type FVB/N mice. Consistent with earlier in vitro findings, increases in granulocyte/macrophage colony-stimulating factor (GM-CSF) and TGF-alpha mRNA transcripts were found in the epidermis at clinically normal sites within 10 weeks after arsenic treatment. Immunohistochemical staining localized TGF-alpha overexpression to the hair follicles. Injection of neutralizing antibodies to GM-CSF after TPA application reduced the number of papillomas in Tg.AC mice. Analysis of gene expression in samples of skin lesions obtained from humans chronically exposed to arsenic via their drinking water also showed similar alterations in growth factor expression. Although confirmation will be required in nontransgenic mice, these results suggest that arsenic enhances development of skin neoplasias via the chronic stimulation of keratinocyte-derived growth factors and may be a rare example of a chemical carcinogen that acts as a co-promoter.

A new enzymatic variant of Leishmania (Leishmania) forattinii isolated from Proechimys iheringi (Rodentia, Echimydae) in Espírito Santo, Brazil.

Taxonomic studies of leishmanial isolates fromthe New World indicate tremendous diversitywithin this genus (E Cupolillo et al. 1994 Am JTrop Med Hyg 50: 296-311). A number of newLeishmania species from sylvan areas of theNeotropics have been described recently. Some ofthose parasites are associated with disease in hu-mans; others appear to be restricted to lower or-ders of mammals, such as rodents and edentates(G Grimaldi et al. 1989 Am J Trop Med Hyg 41:687-725). However, some of the latter parasitesmay yet be shown capable of causing human dis-ease, particularly in persons with altered cellularimmune responses.During a survey of potential reservoir hosts ofLeishmania done in the State of Espirito Santo, Bra-zil (1982-1985), three leishmanial parasites (des-ignated with stock codes MPRO/BR/82/RV203,MPRO/BR/82/RV228, and MPRO/BR/85/RV260)were isolated from skin lesion samples taken fromspiny-rats, Proechimys iheringi (Rodentia,Echimydae), captured in a secondary forest locatedin the municipality of Viana (A Falqueto et al. 1985Mem Inst Oswaldo Cruz 80: 497). In studies us-ing an indirect radioimmune binding assay (RIA)and a large panel of monoclonal antibodies (Mabs)derived for selected species of Leishmania , the iso-lates were characterized as members of the L.mexicana complex. These strains were differentfrom those species infective to humans, in that theyreacted with either the L.(L.) amazonensis or L.(L.) mexicana specific MAbs. Furthermore, thiswas also the case with cloned organisms from twostocks showing that they were not mixed popula-tions of Leishmania (G Grimaldi et al. 1987 Am JTrop Med Hyg 36: 270-287). This group of dis-tinct parasites presented, however, similarserodeme patterns in relation to the new recentlydescribed species of the Leishmania subgenus, L.(L.) forattinii (E Yoshida et al. 1993 Mem InstOswaldo Cruz 88: 397-406).The latter parasite was isolated from sylvaticreservoir hosts (Didelphis marsupialis aurita andP. iheringi denigratus ) captured on regions endemicfor American cutaneous leishmaniasis in Brazil,respectively in the municipalities of Conchas, SaoPaulo (E Yoshida et al. 1979 Rev Inst Med Trop SPaulo 21: 110-113) and Tres Bracos, Bahia (ABarreto et al. 1985 Rev Soc Bras Med Trop 18:243-246). The general morphology and the growthcharacteristics in vitro of the new species were simi-lar to those of other L. mexicana complex para-sites. Here we have further studied the Espirito Santoisolates using additional techniques for the char-acterization of Leishmania . The molecular proce-dures used for typing the strains (isoenzyme elec-trophoresis, blot enzyme binding assay usingMAbs, restriction endonuclease fragment patternsof k-DNA, and molecular karyotype analysis) havebeen described in detail in previous publications(Yoshida et al. 1979, 1983 loc. cit. , Cupolillo et al.1994 loc. cit., A Franco et al. 1997 Mem InstOswaldo Cruz 92: 63-68).Results of electrophoretic analysis were basedon the following enzymatic loci, namely: glucose-6-phosphate dehydrogenase (G6PDH), malate de-hydrogenase (MDH), isocitrate dehydrogenase(IDHNADP), 6-phosphogluconate dehydrogenase(6PGDH), nucleotidase (NH1 and NH2), glucosephosphate isomerase (GPI), phosphoglucomutase(PGM), proline dipeptidase (PEPD), leucine pep-tidase (PEP2), malic enzyme (ME), mannose phos-phate isomerase (MPI), and aconitate hydratase(ACON). The enzyme profiles of the selected iso-lated signature MPRO/BR/82/RV228, which rep-resents the poorly defined group of parasites from

Cutaneous T-Cell Infiltrates: Analysis of T-Cell Receptor γ Gene Rearrangement by Polymerase Chain Reaction and Denaturing Gradient Gel Electrophoresis

In cutaneous T-cell infiltrates, the demonstration of a clonal T-cell receptor (TCR) gene rearrangement has been considered helpful to distinguish Cutaneous T-cell lymphomas from reactive lymphoproliferation. Hence, a polymerase chain reaction (PCR) method using GC-clamp primers and denaturing gradient gel electrophoresis has been developed in our laboratory to analyze the TCR gamma locus configuration. Two hundred eleven cutaneous samples from 155 patients were analyzed. A detectable clonal TCR gamma rearrangement was significantly associated with cutaneous T-cell lymphomas as defined by morphologic and immunologic criteria. A clonal TCR gamma rearrangement was also detected frequently in lymphomatoid papulosis, never in reactive lymphocytic infiltrates and B-cell lymphomas, and rarely in parapsoriasis en plaque and cutaneous lymphoid hyperplasia. Forty five patients had both a cutaneous and a peripheral blood sample. Fifteen had a detectable clonal rearrangement in the two samples and 22 were negative. Six patients had a positive skin sample and a negative blood sample, whereas two patients had a positive blood sample and a negative skin sample. Four lymph node samples were analyzed and the PCR results were the same as in the skin. Finally, 21 patients had sequential samples of recurrent skin lesions. The PCR results were concordant in all and, when detectable, the clonal TCR gamma rearrangement remained unchanged in a given patient. Because of its simplicity and accuracy, the newly designed PCR procedure improves the monitoring of diagnosis, staging, and follow-up in cutaneous T-cell infiltrates.