Florian Butsch

Vaccination with TAT-Antigen Fusion Protein Induces Protective, CD8+ T Cell-Mediated Immunity Against Leishmania Major

In murine leishmaniasis, healing is mediated by IFN-γ-producing CD4(+) and CD8(+) T cells. Thus, an efficacious vaccine should induce Th1 and Tc1 cells. Dendritic cells (DCs) pulsed with exogenous proteins primarily induce strong CD4-dependent immunity; induction of CD8 responses has proven to be difficult. We evaluated the immunogenicity of fusion proteins comprising the protein transduction domain of HIV-1 TAT and the Leishmania antigen LACK (Leishmania homolog of receptors for activated C kinase), as TAT-fusion proteins facilitate major histocompatibility complex class I-dependent antigen presentation. In vitro, TAT-LACK-pulsed DCs induced stronger proliferation of Leishmania-specific CD8(+) T cells compared with DCs incubated with LACK alone. Vaccination with TAT-LACK-pulsed DCs or fusion proteins plus adjuvant in vivo significantly improved disease outcome in Leishmania major-infected mice and was superior to vaccination with DCs treated with LACK alone. Vaccination with DC+TAT-LACK resulted in stronger proliferation of CD8(+) T cells when compared with immunization with DC+LACK. Upon depletion of CD4(+) or CD8(+) T cells, TAT-LACK-mediated protection was lost. TAT-LACK-pulsed IL-12p40-deficient DCs did not promote protection in vivo. In summary, these data show that TAT-fusion proteins are superior in activating Leishmania-specific Tc1 cells when compared with antigen alone and suggest that IL-12-dependent preferential induction of antigen-specific CD8(+) cells promotes significant protection against this important human pathogen.

Bilateral indolent epidermotropic CD8-positive lymphoid proliferations of the ear.

Dear Editors, Reported first by Petrella et al. in 2007 [1], 12 case reports have been published in the literature about slowly growing tumors of the ear histologically termed CD8positive non-epidermotropic lymphomas [2, 3, 4]. Clinically, these lesions, also referred to as “indolent lymphoid proliferation of the ear”, take a benign course, fatalities and systemic spread have not yet been reported, while local recurrences after therapy have been documented [2]. In the following case, for the first time epidermotropism was observed. The patient presented in December 2010 at 73 years of age. He reported a painful nodule on the right ear for three years. A similar lesion on the left ear had receded spontaneously. Clinical examination revealed a red-brown peasized nodule on the helix of the right ear (Figure 1a). A slight erythematous plaque was seen on the opposite helix. Biopsies were performed on both ears. In an identical fashion in both biopsies, the entire dermis along with available subcutaneous fatty tissue was filled by an infiltrate consisting of monomorphic medium-sized lymphocytes with quite a few mitoses. Focally the cells migrated into the basal cell layers of the epidermis (Figure 1b). Immunohistochemically they were CD3-positive, CD8-positive and CD4-negative (Figure 1c). Further, the cells were CD20-negative, CD30negative and CD56-negative with a proliferation index of about 10 % in staining for MIB-1/Ki67. PCR examination for T-cell receptor clonality revealed a clonal band of 186nt as well as a subband of 207nt without a polyclonal background. Molecular pathological testing for Borrelia burgdorferi/ afzelii (OspA gene) and Epstein-Barr virus (EBV, HHV4, EBNA-1 protein gene) demonstrated no specific DNA segments. Computerized tomography of thorax and abdomen as well as PET-CT during the course and after 3 months was normal, excluding extracutaneous manifestations. Differential blood count remained unaltered; the CD4/CD8 ratio was 3. Borrelia serology was normal twice. Radiation therapy was performed with a total dose of 50 Gy – relatively high for a cutaneous lymphoma. After three months total remission was seen. The lesions on the ears of our 73-yearold patient represent a primary cutaneous CD3-positive, CD4-negative, CD8-positive, CD20-negative, CD56negative T-cell lymphoma. The slowly progressive course over 3 years with parBilateral indolent epidermotropic CD8-positive lymphoid proliferations of the ear

Early diagnosis of intravascular large B‐cell lymphoma

In 1959, Pfleger and Tappeiner described a disorder they referred to as “systematized endotheliomatosis of the cutaneous blood vessels” [1]. Despite extensive histomorphological studies, at the time the authors were unable to differentiate whether the lesion represented an unusual carcinoma or hemangioendothelioma. Only many years later was this disorder characterized as intravascular large B-cell lymphoma (IVLBCL) [2]. Since the last decade of the 20th century, the WHO lymphoma classification has listed IVLBCL as subtype of diffuse large B-cell lymphoma (DLBCL) [3]. Herein, we present a case of this very rare disease. In May 2014, the 76-year old female patient first presented to our clinic with a one-year history of erythematous lesions on her legs, which at times gave rise to a burning sensation. She denied recurrent episodes of fever, night sweats, and weight loss. Clinical examination revealed – occasionally bizarrely shaped – livid patches on both legs (Figure 1). Histology showed large atypical cells with pleomorphic nuclei located inside dilated vessel lumina throughout the entire dermis as well as parts of the subcutis. Some of the affected vessels were thrombosed (Figure 2a, b). Immunohistochemically, the cell aggregates showed reactivity for CD20, CD79a, and MUM-1; partially, also for bcl2. The proliferation rate – using MiB1/Ki67 – was increased. The neoplastic cells did not label with CD3, CD10, bcl6, and pancytokeratin MNF116. The endothelial markers CD31 and CD34 showed regular staining of blood vessels, and thus emphasized the intravascular location of tumor cells (Figure 3a, b). While extravascular macrophages/histiocytes and some intravascular monocytes were positive for CD68, intravascular tumor cells showed no such reaction. Immunohistochemistry also showed kappa light-chain restriction (in the absence of lambda reactivity). Molecular analysis showed B-cell clonality (B-cell clonality analysis using multiplex PCR). There was no evidence of tumor cells in the cerebrospinal fluid. Histological examination of osseous tissue (bone punch biopsy) showed regular trilinear hematopoiesis without infiltration of pathological cells. Borrelia serology was negative. Imaging studies (cranial MRl, and CT of the neck, thorax, and abdomen) revealed no evidence of extracutaneous disease involvement; the only remarkable findings included hepatic and ovarian cysts as well as cholelithiasis. The liver was slightly enlarged. Lab tests showed elevated levels of total bilirubin (1.51 mg/dL, normal range Figure 1 Bizarrely shaped livid patches on both legs.

Pembrolizumab-induced lichen planus pemphigoides in a patient with metastatic melanoma

Immune checkpoint inhibitors targeting PD-1 (programmed cell death receptor 1) are indicated in the treatment of advanced melanoma. Cutaneous complications represent the most common immune-related adverse events (irAEs) [ 1 ] . We present the case of an advanced melanoma patient with complete response to treatment with the anti-PD-1 antibody pembrolizumab who developed lichen planus pemphigoides (LPP). A 64-year-old Caucasian man with metastatic melanoma was enrolled in the MK-3475-002 phase II trial comparing pembrolizumab 2 mg/kg or 10 mg/kg (randomized and blinded for the dose received) with standard chemotherapy. Tumor staging showed a partial response after nine cycles of pembrolizumab (given at three-week intervals). However, a CTCAE (Common Terminology Criteria for Adverse Events) grade 2 bullous pemphigoid (BP)-like drug reaction with vesicular lesions of the oral mucosa (Figure 1 a) and scattered papules with central vesicles on the skin prompted the patients exclusion from the trial. Subsequent treatment with topical clobetasol, prednisolone (1 mg/kg PO, followed by a slow taper over the course of four months), and rituximab (375 mg/m2 IV every four weeks; discontinuation after three doses due to CTCAE grade 3 thrombocytopenia) only led to minor improvement of the skin lesions. Six months later, the patient presented with a complete tumor response but also with CTCAE grade 3 white reticular lesions of the oral mucosa ( Wickham striae , Figure 1 b) and pruritic erythematous papules and plaques with central vesicles on the trunk and extremities (Figure 1 c). Histology (biopsy taken from the back) was consistent with lichen planus (LP), showing orthohyperkeratosis, hypergranulosis, cytoid bodies, and lichenoid interface dermatitis with a band-like lymphocytic infi ltrate obscuring the dermoepidermal junction (Figure 1 d, e). Repeated treatment with topical clobetasol and prolonged therapeutic attempts with systemic prednisolone (1 mg/kg PO, followed by a gradual taper over the course of eight months), PUVA therapy (methoxsalen 60 mg PO and UVA 1,2 J/cm2 four times weekly for three weeks), acitretin (50 mg PO once a day for one week), and sirolimus (2 mg PO once a day, discontinuation after eight weeks due to CTCAE grade 3 heart failure) were unsatisfactory.

Topical treatment with a two-component gel releasing nitric oxide cures C57BL/6 mice from cutaneous leishmaniasis caused by Leishmania major.

1. VanGuilder HD, Vrana KE, Freeman WM, Biotechniques. 2008;44:619–626. 2. Eisenberg E, Levanon EY, Trends Genet. 2013;29:569–574. 3. Suzuki T, Higgins PJ, Crawford DR, Biotechniques. 2000;29:332–337. 4. Tricarico C, Pinzani P, Bianchi S, et al. Anal Biochem. 2002;309:293–300. 5. Zainuddin A, Chua KH, Abdul Rahim N, et al. BMC Mol Biol. 2010;11:59. 6. Schmittgen TD, Zakrajsek BA, J Biochem Biophys Methods. 2000;46:69–81. 7. Kheirelseid EA, Chang KH, Newell J, et al. BMC Mol Biol. 2010;11:12. 8. Purmonen S, Manninen T, Pennanen P, et al. Oncol Rep. 2008;19:1627– 1634. 9. Ashcroft KJ, Syed F, Bayat A, PLoS ONE. 2013;8:e75600.

Frühzeitige Diagnose des intravaskulären großzelligen B‐Zell‐Lymphoms

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