The molecular fingerprint of human papillomavirus infection and its effect on the Langerhans cell population in squamous cell carcinomas of the genital skin.

BACKGROUND: Information is scarce about the presence of molecular alterations related to human papillomavirus (HPV) infection in squamous cell carcinomas of the genital skin and about the effect of this infection in the number of Langerhans cells present in these tumors. AIMS: To determine the presence of HPV in genital skin squamous cell carcinomas and to see the relationship between HPV infection and changes in the expression of Ki-67 antigen (Ki-67), p53 protein (p53), retinoblastoma protein (pRb) and E-cadherin and to alterations in Langerhans cell density, if any. METHODS: A descriptive, comparative, retrospective and cross-sectional study was performed with all the cases diagnosed as squamous cell carcinomas of the genital skin at the Dermatopathology Service from 2001 to 2011. The diagnosis was verified by histopathological examination. The presence of HPV was examined using chromogenic in situ hybridization, and protein expression was studied via immunohistochemical analysis. RESULTS: The 34 cases studied were verified as squamous cell carcinomas and 44.1% were HPV positive. The degree of expression of pRb was 17.50% ±14.11% (mean ± SD) in HPV-positive cases and 29.74% ±20.38% in HPV-negative cases (P = 0.0236). The degree of expression of Ki-67 was 47.67% ±30.64% in HPV-positive cases and 29.87% ±15.95% in HPV-negative cases (P = 0.0273). CONCLUSION: HPV infection was related to lower pRb expression and higher Ki-67 expression in comparison with HPV negative samples. We could not find a relationship between HPV infection and the degree of expression of p53 and E-cadherin or with Langerhans cell density.

Increased Langerhans cell accumulation after mycobacterial stimuli.

AIMS: To evaluate the role of Langerhans cells (LCs) in the local activation of leprosy lesions. LCs, acting as tolerance inducers and immune stimuli, are dendritic cells recently implicated in cutaneous homeostasis. The role of LCs in the defence against mycobacterial infection remains poorly understood. METHODS AND RESULTS: The number and distribution of CD1a+ skin cells and HLA-DR and intercellular adhesion molecule (ICAM)-1 expression were analysed in leprosy skin lesions and in delayed-type hypersensitivity (DTH) tests. The results showed a high number of LCs in tuberculin and lepromin tests, in tuberculoid lesions and in the epidermis and dermis during type I and II reactions. In multibacillary lesions, however, the number of LCs was consistently low in comparison with other groups. Increased numbers of LCs were accompanied by marked HLA-DR and ICAM-1 expression, suggesting a strong relationship between these immunological events. CONCLUSIONS: CD1a+ cells are implicated in the local immunological events taking place after mycobacterial stimuli and may account for the local activation of all types of reactional episodes in leprosy.

Langerhans cell histiocytosis of skin: a clinicopathologic analysis of five cases.

BACKGROUND AND AIMS: Langerhans cell histiocytosis (LCH) is a rare proliferative disorder of histiocytes characterized by a proliferation of abnormal and clonal Langerhans cells. We retrospectively studied clinicopathologic features of this disorder in five cases. METHODS: Clinical and histopathological findings of five cases of cutaneous LCH were reviewed based on the hospital records. RESULTS: The age of patients ranged from 28 days to 5 years and M: F ratio was 1:1.5. Clinically, the diagnoses suggested were histiocytosis, varicella, transient neonatal pustular melanosis, keloid, sarcoidosis, seborrheic keratosis and LCH. The most common type of skin lesion was a generalized papular lesion. Histologically, all cases showed aggregates of large mononuclear histiocytes (Langerhans cells) with reniform, irregular, cleaved nuclei and abundant eosinophilic cytoplasm. There was multi-systemic involvement in two patients and single-system involvement in three patients. CONCLUSION: Cutaneous lesions may be the sole presenting feature of LCH. Diagnosis is based on demonstration of S-100 positive histiocytes.

Mycobacterium leprae DNA content, cellular and cytokine patterns in skin lesions of leprosy patients undergoing multidrug therapy (MDT).

Skin biopsies from untreated and MDT-treated patients were examined for infiltrating cells and cells producing the cytokines TNF-alpha, IFN-gamma, and IL-1 beta using immunohistochemistry. Biopsy specimens from untreated tuberculoid leprosy patients were characterized by the presence of cells producing TNF-alpha, IFN-gamma, and IL-1 beta and of subepidermal Langerhans cells. These cells were rarely found or completely absent in biopsies of untreated lepromatous leprosy patients, but tended to increase under MDT. In a short-term therapy trial for three months with brodimoprim, dapsone, and rifampicin, 12 patients were monitored by follow-up biopsies. Semiquantitative PCR for mycobacterial DNA revealed two groups of patients: one group in which mycobacterial DNA in follow-up biopsies remained constant and a second group in which a decrease of mycobacterial DNA during therapy was noted. Immunophenotyping in these follow-up biopsies revealed that in the latter group IFN-gamma-positive cells and Langerhans cells were present and gamma delta T cell receptor-positive cells tended to decrease during therapy. In contrast, in patients whose mycobacterial DNA did not change during therapy, these phenotypical manifestations were not observed. We therefore, conclude that assessment of mycobacterial DNA in combination with phenotyping of infiltrating cells and determination of cytokine patterns may be useful tools in establishing criteria for the effectiveness and duration of MDT in patients with leprosy.

Reduction in CD1 positive epidermal Langerhans cell numbers in leprosy lesions following epicutaneous application of 2:4 dinitrochlorobenzene.

A study was made on Langerhans cells (LC) in the dermal lesions of leprosy after epicutaneous application of 2:4 dinitrochlorobenzene (DNCB) to the lesion. LC were quantitated with OKT6 monoclonal antibody and indirect immunofluorescence. A depletion or reduction in the numbers of CD1+ epidermal LC was observed at both 4 and 24 hours after the application of DNCB in the lesions of both tuberculoid and lepromatous leprosy, compared to untreated lesions.

Two sisters with leprosy.

Leprosy is a rare entity in Japan, but remains quite common in developing countries. We report two sisters with leprosy from Brazil but currently working in Japan who presented to our clinic. The younger sister was infected with the BB type with HLA-A2, A24, B51, Bw52, DR2, DRw8, DRw52, DQw1, and DQw3. The elder sister had the TT type with HLA-A2, A31, B51, Cw3, DRw6, DRw8, DRw52, DQw1, and DQw3. Immunohistochemical findings revealed that CD4+, 4B4+ helper/inducer T-cells were dominant in the granulomas of both cases. Bacilli were not detectable in the biopsy specimens. However, Mycobacterium leprae-specific DNA fragments were found in their peripheral blood and biopsy specimens by polymerase chain reaction.

Novel responses of human skin to intradermal recombinant granulocyte/macrophage-colony-stimulating factor: Langerhans cell recruitment, keratinocyte growth, and enhanced wound healing.

Recombinant granulocyte/macrophage-colony-stimulating factor (rGM-CSF), prepared from Chinese hamster ovary (CHO) cells and Escherichia coli, was administered to 35 patients with the borderline and polar lepromatous forms of leprosy by the intradermal and subcutaneous routes at doses of 7.5-45.0 micrograms/d for 10 d. With each of these doses and routes, increases in the number of circulating eosinophils were noted. After the intradermal injection, the local skin sites demonstrated zones of roughening and micronodularity that appeared within 24-48 h and persisted for more than 6 d. Reinjection of sites led to enhanced areas of epidermal reaction. GM-CSF prepared from CHO cells was a more potent inducer of this effect. GM-CSF given by the subcutaneous route, at higher doses, failed to initiate these changes. At the microscopic level, the epidermis became thickened (+75%) with increased numbers and layers of enlarged keratinocytes. These contained increased numbers of ribosomes and prominent nucleoli, and were imbedded in a looser meshwork of the zona Pellucida. The modified keratinocytes remained MHC class II antigen negative throughout the course of the response. A major change in the dermis was the progressive accumulation of CD1+, Birbeck granule-positive cells. These Langerhans were recognizable at 48 h after intradermal injection and reached maximum numbers by 4 d. During this period the number of epidermal Langerhans cells remained relatively constant. No increment in dermal Langerhans cells occurred when GLM-CSF was injected by the subcutaneous route. No appreciable increase in the numbers of T cells and monocytes was noted, and granulocytes and eosinophils were largely present within the dermal microvasculature. 4-mm punch biopsies taken from injected sites and adjacent controls were compared in terms of the rapidity of wound healing. 22 of 26 sites demonstrated more rapid filling and hemostasis, whereas four were equivalent to controls. We conclude that rGM-CSF, when introduced into the skin, leads to enhanced keratinocyte growth, the selective recruitment of Langerhans cells into the dermis, and enhanced wound healing of the prepared site. There was no evidence of an enhanced cell-mediated response to Mycobacterium leprae, and bacillary numbers remained unchanged.

Immunohistologic assessment of cytokine production of infiltrating cells in various forms of leprosy.

The aim of this study was to determine cytokines in human leprosy lesions by means of immunohistologic examination. Cryostat sections of skin biopsies from 57 patients with various forms of leprosy were immunostained according to the APAAP method, using monoclonal antibodies against interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), and, in addition, against CD 1 antigen. Granulomas in biopsies of untreated patients with tuberculoid leprosy showed large amounts of cells positive for IL-1 beta, TNF-alpha, IFN-gamma, and CD 1, whereas no positive signals could be detected in untreated patients with lepromatous leprosy. However, in those biopsies obtained from lepromatous leprosy patients undergoing chemotherapy, positive staining for cytokines as well as subepidermal Langerhans cells increased to a detectable amount. Remarkably, in tuberculoid leprosy patients, the number of IL-1 beta--positive cells did not vary under therapy, while the number of TNF-alpha and IFN-gamma reactive cells decreased. These results suggest that immunohistologic determination of cytokines in combination with the assessment of subepidermal Langerhans cells in human leprosy lesions may be used as a parameter for the patient's status of cell-mediated immunity under chemotherapeutic treatment.

Differential expression of Langerhans cells in the epidermis of patients with leprosy.

Eighteen patients with lepromatous leprosy (LL) showed a significant reduction (P less than 0.001) of Langerhans cells (LC) irrespective of whether the biopsies were obtained from involved (398 +/- 186) or healthy skin (304 +/- 98). The cells showed morphological changes consisting mainly of loss of dendritic processes. Twenty-four controls (age, sex and race matched) had a mean number of LC of 632 +/- 138. In tuberculoid patients (TT) significant differences were observed, depending on the site of biopsy. Nine biopsies from involved skin had 993 +/- 206 LC, whereas 11 from healthy skin had 448 +/- 96 (P less than 0.001). This difference was confirmed in six additional borderline tuberculoid (BT) and TT patients in whom biopsies were simultaneously obtained from involved (973 +/- 179) and uninvolved skin (498 +/- 99). In 10 patients with indeterminate leprosy the LC density did not differ from the control population (630 +/- 261). The expression of LC numbers in BT and TT patients may represent migration of these cells from healthy skin to involved areas or mobilization of a central pool. The low density found in LL patients could interfere with adequate presentation of mycobacterial antigens leading to tolerance. Alternatively the presence of T helper cells in TT infiltrates may produce factors that recruit LC; their absence in LL lesions may account for the decrease in LC expression.

The reconstitution of cell-mediated immunity in the cutaneous lesions of lepromatous leprosy by recombinant interleukin 2.

Human rIL-2 (10-30 micrograms) was injected intradermally into the skin of patients with lepromatous leprosy with high bacillary loads. All patients responded to the lymphokine with local areas of induration that peaked at 24 h and persisted for 4-7 d irrespective of whether the site was "normal skin" or a nodular lesion. Within 24 h there was an extensive emigration of T cells and monocytes into the site. The percentage of the dermis infiltrated by mononuclear cells increased by more than sevenfold, peaking at 4 d and persisting for greater than 15 d. Both CD4+ and CD8+ T cells entered the site. T cells of CD4+ phenotype predominated at 2-7 d but by 11 d, CD8+ cells were predominant. Considerable numbers of T6+ Langerhans' cells appeared in the dermis by 72 h and persisted for 3 wk. By 4 d the thickness of the overlying epidermis had increased twofold, and keratinocytes were expressing MHC class II antigen and the IFN-gamma-induced peptide IP-10. Starting at 48 h, there was an extensive destruction of mononuclear phagocytes that contained structurally intact or fragmented M. leprae observed at the electron microscope level. The organisms, either free or contained within endocytic vacuoles, were discharged into the extracellular space and then reingested by blood-borne monocytes. This was followed by marked reductions in the number of acid-fast organisms in the injected site, evident as early as 4-7 d and more marked at 2-3 wk after injection. 13 of 15 patients exhibited a disposal of acid-fast bacilli ranging from 5- to 1,000-fold with a mean value of approximately 100-fold. The administration of IL-2 leads to the generation of an effective cell-mediated immune response, recapitulating an antigen-driven event and leading to striking local reductions in M. leprae. In comparison with the purified protein derivative of tuberculin reaction, bacilli are cleared more promptly, although emigratory cells persist for a shorter time.

CD1 positive epidermal Langerhans cells in indeterminate leprosy.

Langerhans cells (LC) in the skin lesions of 10 untreated indeterminate leprosy patients were defined by indirect immunofluorescence using monoclonal antibodies. No difference was observed in the numbers and distribution of epidermal LC in the indeterminate lesions and controls. However, the infiltrates of these lesions contained CD1+ cells. Most cells in the infiltrates HLA DR antigens.

Distribution and turnover of Langerhans cells during delayed immune responses in human skin.

The changes in distribution and turnover of T6+ Langerhans cells (LC) in the skin during delayed immune responses to tuberculin, and in the lesions of tuberculoid leprosy and cutaneous Leishmaniasis were investigated. In each situation, there was a dermal accumulation of monocytes and T cells and epidermal thickening with keratinocyte Ia expression. In the tuberculin response a dramatic change in the distribution of LC was observed. By 41 h, T6+ LC were displaced to the upper zone of the thickening epidermis followed by an almost complete loss of LC from the epidermis by approximately 72 h. After 7 d, T6+ cells started to reappear in the epidermis, which regained almost normal numbers of T6+ LC by 14 d. After antigen administration and initiation of the delayed immune response, enhanced numbers of T6+ cells appeared in association with the mononuclear cell infiltrate of the upper dermal lesions. Their numbers peaked by 72 h, were reduced at 7 d, and again enhanced by 14 d, when the epidermis was being repopulated. Similar numbers of T6+ cells were found in the chronic lesions of tuberculoid leprosy and cutaneous Leishmaniasis but not lepromatous leprosy. The cells of the dermis were identified as typical LC by the presence of Birbeck granules and surface T6 antigen at the electron microscope level. These cells were closely associated with lymphocytes. We have quantified the number of LC, evaluated their directional flux into the epidermis and dermis, determined nearest neighbors, and made predictions as to their fate.

OKT6+ epidermal Langerhans' cell numbers in DNCB reactions among leprosy patients.

A study was made on the Langerhans' cells at the sites of contact sensitivity skin reactions in 45 untreated leprosy patients. The skin reaction was induced by 2,4-dinitrochlorobenzene (DNCB). Langerhans' cells were quantitated using OKT6 monoclonal antibody and indirect immunofluorescence. Clinically, the skin reaction in the tuberculoid patients was positive at 4, 24, and 48 hr, while the lepromatous patients failed to respond at any of the time intervals. Sequential histological analysis of the skin reaction showed predominantly mononuclear cell infiltrates around the blood vessels and neurovascular bundles in both the tuberculoid and lepromatous patients. Time kinetic assessment showed no difference in the numbers and distribution of OKT6+ epidermal Langerhans' cells at the site of the DNCB skin reactions among the tuberculoid and lepromatous patients. This, therefore, suggests that either there is a functional defect in Langerhans' cells or some other mechanism(s) such as a T-cell abnormality is responsible for the lack of clinical reaction in lepromatous patients.

Epidermal keratinocyte Ia expression, Langerhans cell hyperplasia and lymphocytic infiltration in skin lesions of leprosy.

Epidermal changes, Ia expression on keratinocytes, Langerhans cell hyperplasia and lymphocyte infiltration were sought in skin lesions of leprosy: 15 borderline tuberculoid (BT), six borderline lepromatous (BL), 17 lepromatous (LL), 13 erythema nodosum leprosum (ENL), six Lucio reactions and nine reversal reactions. All three changes were well developed in BT and reversal reactions. ENL showed well developed keratinocyte Ia and Langerhans cell hyperplasia, but little lymphocytic infiltration. LL and Lucio tissues had some Langerhans cell hyperplasia but little or no keratinocyte Ia or lymphocytic infiltration. BL tissues were so diverse as to suggest two distinct subgroups. These findings are consistent with the hypothesis that keratinocyte Ia expression is an immunohistological sign of a cell-mediated immune (CMI) response. However, the Ia keratinocyte expression found in BL and ENL tissues appears contrary to the undifferentiated macrophages and numerous bacilli found in the lesions. Thus, if a sign of CMI, keratinocyte Ia expression is not a measure of the effectiveness of the response.

Local and systemic effects of intradermal recombinant interferon-gamma in patients with lepromatous leprosy.

Evidence that interferon-gamma may be a physiologic macrophage-activating factor, and that macrophage activation may be defective in lepromatous leprosy, led us to test the effects of intradermal injection of low doses of recombinant interferon-gamma in six patients with this disease. Interferon-gamma, 1 or 10 micrograms, was administered daily by jet gun for three days into a single cutaneous lesion. A biopsy specimen was taken from the injection site on the sixth study day and compared with specimens obtained previously from a site where no injection had been made or where excipient alone had been injected in the same way as the interferon. Interferon-gamma elicited local effects similar to certain features of delayed-type hypersensitivity reactions or tuberculoid leprosy, including induration, T-cell and monocyte infiltration, keratinocyte proliferation, diminution of epidermal Langerhans cells, and dermal and epidermal cell HLA-DR (Ia) antigen expression. At some of the sites of interferon-gamma injection, there was also an apparent decrease in acid-fast bacilli. Before treatment, monocytes from patients with lepromatous leprosy released 48 percent as much hydrogen peroxide as did monocytes from controls in response to phorbol myristate acetate, and 36 percent as much as those from controls in response to Mycobacterium leprae. When recombinant interferon-gamma was injected, these responses became normal. No toxic effects were observed. These observations suggest that interferon-gamma can mediate certain manifestations of delayed-type hypersensitivity or cell-mediated immunity in vivo, and that recombinant interferon-gamma should be tested for possible therapeutic effects in certain nonviral infectious diseases.