In vivo selective expansion of a tumour-specific cytotoxic T-cell clone derived from peripheral blood of a melanoma patient after vaccination with gene-modified autologous tumour cells.

Melanoma-specific cytotoxic T lymphocytes (CTL) can be generated from peripheral blood lymphocytes (PBL) by mixed lymphocyte-tumour cell cultures. Analysis of CTL precursor frequencies in peripheral blood of melanoma patients is generally used for immunomonitoring purposes to evaluate vaccination efficacy. At present, it is unclear whether PBL-derived CTL generated in vitro are indicative of an anti-tumour immune response in vivo. Three tumour-specific human leucocyte antigen (HLA)-B/C-restricted CTL clones were derived from peripheral blood of a melanoma patient immunized with interleukin-7 (IL-7) gene-modified tumour cells. CTL clones differing in their T-cell receptor-gamma (TCRgamma) rearrangement produced interferon-gamma, IL-4 and/or IL-10. On the basis of their unique TCRgamma gene rearrangements clone-specific primers were generated for detection of clone-specific DNA by polymerase chain reaction. One CTL clone (E5) of the three was found to be selectively expanded in one of seven metastases obtained at autopsy, as determined by Southern blot hybridization. However, the presence of E5 in only one of seven metastases at death indicates that the in vivo accumulation of the specific CTL clone was not sufficient to contain tumour progression. Nevertheless, our data support the proposition that analysis of anti-tumour activity of PBL-derived CTLs may reflect an anti-tumour immune response in vivo.

Tumor markers in peripheral blood of patients with malignant melanoma: multimarker RT-PCR versus a luminoimmunometric assay for S-100.

The identification of circulating tumor cells in the peripheral blood of patients with malignant melanoma by detection of melanoma associated protein transcripts using the reverse transcriptase polymerase chain reaction (RT-PCR) technique has been introduced as a noninvasive and sensitive technique for early detection of tumor progression and metastatic disease. An alternative approach is the analysis of S-100 protein in the serum of melanoma patients by a luminoimmunometric assay (LIA). In this study, the sensitivities of RT-PCR and LIA were compared. Seventy-seven blood samples of 59 melanoma patients were analyzed for tyrosinase, Melan-A/MART-1, MAGE-3, gp100, and p97 expression by multimarker RT-PCR; 540 serum samples of 352 melanoma patients were analyzed for S-100 protein concentration by LIA. In stage III 23.8% and in stage IV 37.5% of the samples were positive for at least one marker in multimarker RT-PCR, versus 8.1% and 48.1% of elevated S-100 levels analyzed by LIA, respectively. In a direct comparison, 31 identical samples were analyzed by multimarker RT-PCR and by S-100 LIA. In stage III 18.2% and in stage IV 45% of the samples were positive by multimarker RT-PCR versus 45.5% and 80% by S-100 LIA, respectively. S-100 LIA was more sensitive in detection of metastatic disease in melanoma patients than multimarker RT-PCR and should be evaluated in further studies. RT-PCR might be more useful in the analysis of micrometastases in anatomic compartments other than peripheral blood.

Molecular diagnosis of deep nodular bacillary angiomatosis and monitoring of therapeutic success.

A 51-year-old human immunodeficiency virus (HIV)-positive male patient (CDC stage 3C) had had a painful nodule on his external ankle joint for 10 months. A biopsy suggested bacillary angiomatosis, but Kaposi's sarcoma could not be excluded. Rods were detectable in lesional skin by a Warthin-Starry stain. A 298 base pair (bp) gene fragment specific for Bartonella species was amplified from lesional skin and direct nucleotide sequence analysis of the amplification product clearly identified Bartonella quintana. Kaposi's sarcoma-associated herpes virus specific DNA was not amplifiable by polymerase chain reaction (PCR) in our patient, suggesting that the lesion represented bacillary angiomatosis alone, despite clinical and histopathological features which suggested the coexistence of bacillary angiomatosis and Kaposi's sarcoma. The lesion regressed after erythromycin was prescribed. However, 4 and 9 weeks after initiation of therapy, PCR still yielded a positive result in material obtained by a swab. After complete healing, following 12 weeks of antibiotic therapy, PCR became consistently negative. The optimal length of antibiotic treatment in HIV-positive patients with bacillary angiomatosis is not yet known and inadequate therapy may be followed by disseminated disease and a fatal outcome. PCR-based monitoring of the success of treatment is valuable for determining the duration of treatment resulting in a cure.

Detection of herpes simplex virus in exacerbated pemphigus vulgaris by polymerase chain reaction.

BACKGROUND: Herpes simplex virus infections are well known complications of various dermatoses and have also been reported in acantholytic diseases like pemphigus vulgaris or Darier's and Hailey-Hailey diseases. In pemphigus vulgaris, herpes simplex virus infection is considered to be rare and difficult to rule out clinically. OBJECTIVE: We report on 3 patients suffering from pemphigus vulgaris with exacerbation especially of lesions of the oral mucosa. METHODS AND RESULTS: While conventional techniques failed to unequivocally support a suspected herpetic infection, herpes simplex virus-specific DNA was detected by polymerase chain reaction (PCR) in cytological swabs taken from oral erosions of all 3 patients. CONCLUSION: Herpetic infection should be considered in pemphigus vulgaris with lack of improvement under adequate immunosuppressive therapy. In addition, herpes simplex virus infection might to able to induce acute exacerbation of oral pemphigus. PCR can be useful for a highly sensitive and rapid molecular detection of herpes simplex virus.

Molecular evidence for the existence of disseminated zoster as a distinct entity in an immunosuppressed renal transplant patient.

Immunosuppressed renal transplant recipient are at substantially increased risk for the development of varicella zoster virus infections. They are also more prone than immunocompetent patients to develop atypical zoster and to experience a protracted course, and among them there is a higher frequency of generalized infections with possible fatal outcome. While establishing the diagnosis is essential to provide adequate therapy, conventional laboratory methods frequently fail to confirm the suspected infection. We report on a 47-year-old renal transplant recipient who developed multiple necrotic cutaneous ulcers under immunosuppressive treatment. While electron-microscopic analysis (negative staining) revealed no viral structures, varicella zoster virus specific DNA was detected by polymerase chain reaction in material obtained by a swab from these ulcers. Atypical herpetic infection should also be considered as a cause of disseminated ulcerative or necrotic skin lesions in immunosuppressed patients. Assays based on polymerase chain reaction are useful for the rapid confirmation or rejection of the suspected diagnosis of atypical herpetic infection.

[Exacerbation of Hailey-Hailey disease by infection with herpes simplex virus. Detection with polymerase chain reaction]. / Exazerbation eines Morbus Hailey-Hailey durch Infektion mit Herpes-simplex-Virus. Nachweis mittels Polymerasekettenreaktion.

A 49-year-old man suffered from Hailey-Hailey disease for several years. The patient presented with an acute exacerbation of the disease, which did not respond to oral treatment with high doses of glucocorticosteroids. A skin biopsy was taken and the histological examination indicated a viral infection. Herpes simplex virus was confirmed by electron microscopy (negative staining) and polymerase chain reaction (PCR). The PCR presents a sensitive and effective molecular-biological method for the diagnosis of viral infections.

No evidence for Borrelia burgdorferi-specific DNA in lesions of localized scleroderma.

A possible association of Borrelia burgdorferi with localized scleroderma is currently the focus of intense research and discussion. Skin biopsies from 30 patients with localized scleroderma (28 of the plaque type/morphea; two linear scleroderma) were analyzed for the presence of Borrelia burgdorferi using three different polymerase chain reaction systems for amplification of segments of borrelial genes. Formalin-fixed, paraffin-embedded biopsies of 14 patients and fresh-frozen, cryo-conserved biopsies of 16 patients with localized scleroderma were obtained. Lesions of all patients showed clear signs of scleroderma and disease progression at the time of biopsy. Fresh-frozen as well as formalin-fixed biopsies from patients with erythema migrans or acrodermatitis chronica atrophicans were used as positive controls. With all three polymerase chain reaction systems, borrelial DNA was detected in none of the 30 specimens of localized scleroderma. In contrast, with one polymerase chain reaction system, Borrelia burgdorferi-specific DNA was found in 24 of 27 frozen biopsies from patients with erythema migrans and in all 5 analyzed frozen biopsies of patients with acrodermatitis chronica atrophicans. In approximately half of the paraffin-embedded biopsies from patients with erythema migrans (nine of 23) and acrodermatitis chronica atrophicans (13 of 27), Borrelia burgdorferi-specific DNA was identified. These results question the association of localized scleroderma with known subtypes of Borrelia burgdorferi.

Detection of Chlamydia trachomatis in semen by antibody-enzyme immunoassay compared with polymerase chain reaction, antigen-enzyme immunoassay, and urethral cell culture.

OBJECTIVE: To compare the results obtained by four different techniques for the detection of Chlamydia trachomatis in the male genital tract. DESIGN: Prospective study. SETTING: Andrology unit of a university hospital. PATIENTS: Male infertility patients. INTERVENTIONS: Analysis of semen samples and urethral swabs for the presence of C. trachomatis by recombinant antibody-enzyme-linked immunosorbent assay (rELISA), polymerase chain reaction (PCR), antigen-enzyme immunoassay (EIA) and McCoy cell culture. MAIN OUTCOME MEASURE: Detection of C. trachomatis. RESULTS: In 57 of 205 semen samples (27.8%) immunoglobulin A-antibodies against C. trachomatis were found. In contrast, only 1 of 56 semen samples (1.8%) was positive for C. trachomatis-DNA by PCR, only 1 of 139 semen samples (0.7%) was positive by antigen-EIA, and only 4 of 173 urethral swabs (2.3%) grew C. trachomatis in cell culture. CONCLUSIONS: The discrepancy of positive results found by the antibody-rELISA and direct methods for the detection of C. trachomatis indicates successful eradication of the microorganism in > 90% of antibody-positive men. Therefore, detection of antibodies against C. trachomatis in seminal plasma appears to be of limited diagnostic value.

Molecular subtyping of Borrelia burgdorferi in erythema migrans and acrodermatitis chronica atrophicans.

Recently, three subtypes of Borrelia burgdorferi have been identified: Borrelia burgdorferi sensu stricto, Borrelia garinii, and the VS 461 group of Borrelia burgdorferi. These subtypes differ by nucleotide sequence variations within several Borrelia burgdorferi specific genes and most likely by their pathogenetic potential. To assess whether different subtypes of Borrelia burgdorferi might be associated with different cutaneous manifestations and clinical courses of Lyme disease, lesional skin biopsies from 35 patients with erythema migrans and 18 patients with acrodermatitis chronica atrophicans were analyzed. A Borrelia burgdorferi specific gene segment encoding a 26-kD protein with subtype specific nucleotide sequence variations was amplified by a nested polymerase chain reaction technique. For molecular subtyping, the products were transcribed into complementary RNA. Upon polyacrylamide gel electrophoresis, complementary RNA molecules separate into several metastable conformational forms resulting in patterns of bands highly specific for the nucleotide sequence of the transcribed molecules. In biopsy specimens of erythema migrans, the VS 461 subtype was detected in 28 of 35 and the Borrelia garinii subtype in six of 35 cases. In one of 35 cases of erythema migrans Borrelia burgdorferi sensu stricto as well as Borrelia garinii was detected. In contrast, in all 18 biopsies of acrodermatitis chronica atrophicans, only the VS 461 subtype was identified. This subtype is rarely found in the USA, where acrodermatitis chronica atrophicans is almost unknown. These data indicate that acrodermatitis chronica atrophicans might be closely associated with the VS 461 group of Borrelia burgdorferi.