Allogeneic gene-modified tumor cells (RCC-26/IL-7/CD80) as a vaccine in patients with metastatic renal cell cancer: a clinical phase-I study.

Despite novel targeted agents, prognosis of metastatic renal cell cancer (RCC) remains poor, and experimental therapeutic strategies are warranted. Transfection of tumor cells with co-stimulatory molecules and/or cytokines is able to increase immunogenicity. Therefore, in our clinical study, 10 human leukocyte antigen (HLA)-A(*)0201(+) patients with histologically-confirmed progressive metastatic clear cell RCC were immunized repetitively over 22 weeks with 2.5-40 × 10(6) interleukin (IL)-7/CD80 cotransfected allogeneic HLA-A(*)0201(+) tumor cells (RCC26/IL-7/CD80). Endpoints of the study were feasibility, safety, immunological and clinical responses. Vaccination was feasible and safe. In all, 50% of the patients showed stable disease throughout the study; the median time to progression was 18 weeks. However, vaccination with allogeneic RCC26/IL-7/CD80 tumor cells was not able to induce TH1-polarized immune responses. A TH2 cytokine profile with increasing amounts of antigen-specific IL-10 secretion was observed in most of the responding patients. Interferon-γ secretion by patient lymphocytes upon antigen-specific and non-specific stimulation was substantially impaired, both before and during vaccination, as compared with healthy controls. This is possibly due to profound tumor-induced immunosuppression, which may prevent induction of antitumor immune responses by the gene-modified vaccine. Vaccination in minimal residual disease with concurrent depletion of regulatory cells might be one strategy to overcome this limitation.

Diagnostic tools in mycosis fungoides.

Cutaneous T-cell lymphomas (CTCL) represent clonal proliferations of neoplastic skin homing T-cells. Within the group of primary CTCL, mycosis fungoides (MF) is the most common entity, affecting the skin as a primary site. MF initially presents in the skin with a slow indolent course of a characteristic stepwise progression from patches to plaques and tumors accompanied by distinctive histological changes. Routine diagnosis is based on these clinical and histological features. However, due to similarities with benign lymphoproliferative or reactive skin diseases, especially at the initial presentation of the disease, diagnosis can be difficult. Although the etiology of mycosis fungoides is still unknown, important insights have been gained in the immunological and genetic perturbations, which are associated with the disease. In the last years the emergence of molecular genetic techniques allowing to analyze the clonality status in lymphocytic infiltrates, has provided new tools with the potential to increase the accuracy of diagnosis, staging and therefore stage-adapted treatment. Nevertheless, it is important to notice that some limitations restrict the predictive value of the results obtained by these analyses. Diagnostic tool of MF, including clinical, histo- and immunohistological findings as well as molecular genetic analysis will be covered in this review.

Detection of expanded T cell clones in skin biopsy samples of patients with lichen sclerosus et atrophicus by T cell receptor-gamma polymerase chain reaction assays.

Lichen sclerosus et atrophicus is a chronic dermatosis of unknown etiology and pathogenesis. Lichen sclerosus et atrophicus associated skin lesions show T cell enriched infiltrates, sometimes resembling the histologic picture of early mycosis fungoides. It is supposed that the infiltrating T cells participate in the pathogenesis of atrophy and sclerosis. We investigated skin biopsies from 39 lichen sclerosus et atrophicus patients by histology, immunohistochemistry and, in order to establish the status of T cell clonality, by polymerase chain reaction amplifying the T cell receptor-gamma rearrangements. A stage-dependent shift of the CD3-positive T cells was observed from a predominantly CD4-positive to a predominantly CD8-positive phenotype. The increase of CD8-positive cells was associated with more pronounced epidermotropism and basal degeneration. Nearly all CD8-positive cells expressed cytotoxic granules (TIA1), possibly causing the basal destruction. In the late fibrotic stage of the disease, only a weak or no infiltrate was found. Regarding the T cell receptor-gamma polymerase chain reaction, the presence of clonally expanded T cells was demonstrated in 19 of 39 patients (49%) by at least one of two different high resolution electrophoresis techniques applied to separate the amplification products. Thus, for the first time clonally expanded infiltrating T cells were detected in lichen sclerosus et atrophicus. Furthermore, this is one of the first reports on the detection of clonally expanded infiltrating T cells in an inflammatory skin disease. The clonal T cells could not be assigned to the CD4 or CD8 subtype. Most likely, their presence is not the result of a malignant transformation but a response to an as yet unknown lichen sclerosus et atrophicus associated antigen.

Detection of melanoma micrometastases in the sentinel lymph node and in nonsentinel nodes by tyrosinase polymerase chain reaction.

The aim of our study was to investigate the metastatic pathways of melanoma cells in sentinel and other regional lymph nodes. The term "sentinel lymph node" means that the first lymph node of the draining site of a primary tumor is never bypassed in malignant melanoma. In this case lymph node dissection would be necessary only when melanoma cells are detected in the sentinel node. Tyrosinase reverse transcriptase-polymerase chain reaction was applied to search for metastatic melanoma in the sentinel lymph node and in further lymph nodes of a complete lymph node basin in patients who underwent lymph node dissection. In 24 patients with malignant melanoma the draining site of the tumor was marked by lymphoscintigraphy and by intraoperative injection of patent blue V in the area around the primary tumor. The lymph nodes of the affected basin were excised and prepared for histopathologic, immunohistochemical, and molecular biologic examinations. Regarding the sentinel lymph node, 10 of 24 patients showed morphologic evidence for metastases, three additional patients showed only tyrosinase transcripts. In 11 of these 13 cases we found one or more nonsentinel lymph nodes with morphologically detectable melanoma cells and/or tyrosinase mRNA. Interestingly, in seven of 24 patients a positive tyrosinase reverse transcriptase-polymerase chain reaction was received in nonsentinel lymph nodes, whereas the sentinel lymph node was negative, not only for all histologic examinations but also by tyrosinase reverse transcriptase-polymerase chain reaction. In five of seven patients of the latter group, gp100 reverse transcriptase-polymerase chain reaction was carried out, showing also gp100 mRNA in nonsentinel lymph nodes only. Our data indicate that the concept of the sentinel lymph node may miss micrometastases. Whether such micrometastases cause a recurrence or a metastasis of malignant melanoma, or can be destroyed by the immune system, remains to be clarified.

Microanatomical compartments of clonal and reactive T cells in mycosis fungoides: molecular demonstration by single cell polymerase chain reaction of T cell receptor gene rearrangements.

Mycosis fungoides (MF) is a cutaneous T cell lymphoma, clinically characterized by patches, plaques and tumors occurring in successive stages of the disease. In early MF, an infiltrate consisting of mainly reactive T cells is seen in the papillary dermis while tumor cells are mostly confined to the epidermis. By contrast, later stages show nodular infiltrates formed mostly of tumor cells in the dermis while the epidermis is relatively devoid of tumor cells; however, knowledge of the localization of clonal T cells has been based on histomorphologic features and immunohistochemical stainings visualizing certain V-beta subfamilies of the T cell receptor (TCR). As these techniques do not allow for an unequivocal identification of clonal tumor cells, we used micromanipulation and single cell PCR amplifying the TCR chain gene rearrangement. A total number of 387 single T cells was isolated from six skin biopsies in five patients in patch, plaque, and tumor stages. Of these, 180 T cells were picked from the epidermis and 207 from the dermal infiltrate. The rearranged TCR-gamma DNA could be sequenced from 181 of 387 T cells. In three of six patients representing all three stages, epidermal T cells with a clonal rearrangement could be amplified. In early plaque stage a higher degree of epidermal T lymphocytes was found than in initial patch, later plaque, and tumor stages with an inverse distribution found for reactive T lymphocytes. In two patients a biallelic rearrangement was demonstrated that had not been detected in prior PCR analysis from blood and skin samples. These data show that clonal (neoplastic) and non-clonal (reactive) T lymphocytes in MF preferentially infiltrate different microanatomical compartments of the skin, depending on the stage of disease. The microanatomically distinct localization of reactive and clonal T cells suggests that the absence of direct contact between tumor and host-defense lymphocytes may contribute to tumor persistence and progression in epidermis, peripheral blood, and deep dermal tumor cell nests, respectively.

An enzyme-linked immunosorbent assay for the quantification of solubilized CD14 in biological fluids.

A simple and robust two site-binding ELISA for the quantification of solubilized CD14 in human and animal body fluids is described. The principle of the assay depends on the specific binding of sCD14 to two monoclonal antibodies (MEM-18, RoMo-1) recognizing different epitopes of this glycoprotein. The detection limit for sCD14 was 1 ng/ml. The method was used to quantify sCD14 in different biological fluids, giving an intra-assay coefficient of variation and an interassay coefficient of variation of about 9%. The assay was used to measure sCD14 in human serum and plasma and other body fluids in health and disease, and in cell culture supernatants. With the exception of monkeys there was no reactivity with 29 other species screened. In healthy volunteers the sCD14 serum level had a mean value of 3.98 +/- 0.3 micrograms/ml (mean SEM, n = 102).

[The role of fibronectin in nonspecific defense from the immunologic viewpoint]. / Zur Rolle von Fibronektin bei der unspezifischen Abwehr aus immunologischer Sicht.

The present article gives a review of the essential data of literature concerning the importance of fibronectin (Fn) for the unspecific defence. In this case the function of Fn postulated as unspecific opsonin stands in the foreground. Fn mediates the binding of gelatine particles, bacteria and other above all particular materials on phagocytizing cells (monocytes, macrophages and neutrophils). Up to now it is, however, controversial whether or not this binding causes an increased endocytosis. The participation of cofactors or inhibitors and the problem whether Fn or certain proteolytic Fn-fragments are effective still need a further clarification. Fn, its fragments or Fn-substrate-complexes can activate phagocytes and have a chemotactic effect on them. The mechanism of activation is still unknown, for monocytes in increased expression of Fc- and C3b-receptors is supposed. Fn might consequently perform a defence function as an unspecific cell activator and at the same time have a pro-inflammatory influence. There are controversial opinions as to the questions which phagocytes are activated and which Fn-molecules (complete plasma-Fn or fragments) are responsible. The high sensitiveness of Fn to proteolytic enzymes and denaturating processes is supposed to be one of the causes for the different results.

[The diagnostic value of antinuclear antibodies]. / Zur diagnostischen Bedeutung von antinukleären Antikörpern.

In this review we focus on the diagnostic importance of antinuclear antibodies (ANA), the biological function of the relevant autoantigens and on some methodological questions regarding the detection of ANA. The qualitative and quantitative evaluation of ANA has improved significantly in recent years with the introduction of several new test kits. A precondition for the rational use of those assays is knowledge of the diagnostic validity of the detected ANA with regard to the method used. ANA are autoantibodies that react with nucleic acids, protein-nucleic acid complexes and proteins of nuclei. The reasons for their in vivo production are unknown. ANA characterize several of the so-called connective tissue autoimmune diseases and their subtypes, either alone or in typical combinations. They differ significantly with regard to their prevalence and thus in their diagnostic validity. Specificity and prevalence do not correlate. ANA are not markers of disease activity except for antibodies against dsDNA. ANA levels can be interpreted correctly only in connection with clinical symptoms and other laboratory findings.

Preparation of human alpha-2-macroglobulin by chromatography on Cibacron Blue Sepharose in the presence of pregnancy- associated alpha-2-glycoprotein.

The simple and efficient procedure for the isolation of alpha-2-macroglobulin (alpha-2-M) from human sera (hp-type 1-1) by means of affinity chromatography on Cibacron Blue Sepharose is not convenient to separate it from pregnancy-associated alpha-2-glycoprotein (alpha-2-PAG) which is present in high amounts in sera of estrogen-treated women, at pregnancy, and under other conditions. With this method both proteins are eluted in the same fractions; gel filtration of these fractions does not lead to their separation. Therefore, the use of male sera (tesed by monospecific antisera to alpha-2-PAG) with low alpha-2-PAG, content (hp-type 1-1) is recommended for alpha-2-M preparation.

No inhibition of the lymphocyte transformation by pregnancy-associated alpha 2-glycoprotein (PAAG).

Fractions of retroplacental sera containing pregnancy-associated alpha 2-glycoprotein (PAAG) inhibited the lymphocyte transformation. The inhibitory effect did not correlate to the amount of this protein. It remained even after a specific depletion of the PAAG without altering the composition of the other proteins in the sera fractions. These results rule out a suppressive activity of the PAAG in the lymphocyte transformation.

Search for Kaposi's sarcoma-associated virus DNA in hemangioproliferative disorders and cutaneous malignant lymphoma.

Recently, a Kaposi's sarcoma-associated herpesvirus (KSHV) was discovered. We evaluated by PCR 14 paraffin-embedded specimens with the histological diagnosis of endemic, classic and HIV-associated Kaposi's sarcoma (KS) for the presence of the KSHV DNA sequence. In addition, biopsies of adjacent, histologically unaffected skin, peripheral-blood mononuclear cells (PBMCs) of HIV-infected KS patients, PBMCs of one classic KS patient, and specimens of patients with hemangioproliferative disorders other than KS as well as samples of cutaneous T- and B-cell lymphoma were analyzed for KSHV. In all cases of KS, independent of the KS subtype, KSHV was detected in lesional skin. No KSHV was found in biopsies of the adjacent unaffected skin or PBMCs of HIV-infected KS patients. We found KSHV in the PBMCs of a patient with classical KS. All specimens of cutaneous T- and B-cell lymphomas or lymphomatoid papulosis were negative for KSHV. In addition, the samples with hemangioproliferative disorders other than KS were negative for KSHV. There was one borderline case of KS or acroangiodermatitis that was positive for KSHV. Additional histological sections and clinical evaluation confirmed the diagnosis of classic KS. In summary, the data indicate that PCR for KSHV should be a useful diagnostic tool in cases of hemangioproliferative disorders.

[Structure and function of the immunoglobulin Vh gene in man]. / Aufbau und Funktion der Immunglobulin-Vh-Gene beim Menschen.

The present study provides a survey on the structure and functions of human Vh-genes. The more than 100 individual gene segments on chromosome 14 are classified, according to sequence homologies, into 6 families. They differ very much in size and contain pseudogenes, form an interspersed cluster, exhibit homologies with mouse genes and have phylogenetically developed from a single primordial gene. There seem to be no association of antibody specificities with distinct Vh genes or families. Reports of preferential VH gene usage in the fetal stage or in autoantibody synthesis require confirmation. Vh genes for Ig production in the adult mammalian organism are, most probably randomly selected.

Detection of clonal T cells in cutaneous T cell lymphoma by polymerase chain reaction: comparison of mutation detection enhancement-polyacrylamide gel electrophoresis, temperature gradient gel electrophoresis and fragment analysis of sequencing gels.

Cutaneous T cell lymphomas (CTCL) can be differentiated from benign inflammatory dermatoses by the demonstration of clonal T cells in skin biopsy. As a marker of the T cell clonality, the rearrangement of the T cell receptor (TCR) genes is amplified by polymerase chain reaction (PCR) and subsequently analyzed by several electrophoresis techniques. Since the validity of this approach depends substantially on the separating capacity of the applied electrophoresis technique, we investigated in the present study the lower detection limit and the sensitivity of heteroduplex-loaded polyacrylamide gel electrophoresis on MDE (mutation detection enhancement) gels (HD-MDE PAGE), of heteroduplex-loaded temperature gradient gel electrophoresis (HD-TGGE) and fragment analysis (FA) on sequencing gels. Genomic DNA from formalin-fixed, paraffin-embedded skin biopsies of 53 CTCL specimens and 27 samples of benign dermatoses was analyzed by TCRy PCR followed by electrophoretic separation. Clonality was detected by HD-MDE PAGE in 22, by HD-TGGE in 34, and by FA in 33 of the 53 CTCL cases. Additionally, FA revealed an oligoclonal fragment profile in seven CTCL specimens. In the 27 samples from benign dermatoses, HD-MDE PAGE and HD-TGGE showed the expected polyclonal pattern in 26, and FA in 25 specimens. HD-TGGE and FA detected a clonal pattern down to a dilution of 10(3) monoclonal cells in 10(6) peripheral blood mononuclear cells (PBMC), while HD-MDE PAGE revealed a detection limit of 10(4) monoclonal cells in 10(6) PBMC. In conclusion, HD-TGGE and FA possess a higher sensitivity and lower detection limit than HD-MDE PAGE. Therefore, both former techniques are useful tools for the routine diagnostic procedure. With regard to time and cost, we recommend HD-TGGE.

[Fibronectin concentrations in the plasma of newborn infants and their mothers and various plasma preparations]. / Fibronektinkonzentrationen im Plasma von Neugeborenen und deren Müttern sowie verschiedenen Plasmaprärationen.

The article describes a method for the quantification of plasma fibronectin (FN) by means of electroimmunodiffusion after Laurell. By this means the fibronectin concentrations of healthy, mature newborns as well as of their mothers were determined. The average FN-level of the newborns lies at 33% related to the value for adults and at 48%, respectively, related to the maternal FN-content. There is no difference between male and female newborns. The FN-concentrations of the adults stated by us with 330 and 314 mg/l, respectively, for males and females correspond to the data in literature. Furthermore, the FN-content in plasma preparations was determined, with an average value of 2,356 mg/l this was highest in the factor-VIII-concentrate. The expediency and the prerequisites for a possible fibronectin substitution are discussed.

Development of a solid phase enzyme immunoassay for the detection of anti-Ro autoantibodies.

An enzyme immunoassay was developed to detect anti-Ro(SS-A) autoantibodies. Both Ro-antigen components (52 and 60 kD) were purified from a pig spleen extract, using fast protein liquid chromatography (FPLC). Anti-La, anti-RNP, anti-DNA and anti-Sm antibodies do not react to the purified antigen. There was a strong correlation between anti-Ro activity in EIA and the titers in counter immunoelectrophoresis (rs = 0.893). Anti-Ro antibodies were found in 54 (69.2%) of 78 SLE sera by the developed EIA.

[Detection of monoclonal T-cells with TCR gamma PCR in mycosis fungoides]. / Nachweis von monoklonalen T-Zellen mittels TCR gamma-PCR bei Mycosis Fungoides (MF).

The monoclonal dominant malignant T cells in mycosis fungoides (MF) carry identical TCR gamma rearrangements. Their detection is a useful diagnostic tool. Thus, in routine diagnosis we investigated the occurrence of monoclonal T cells in skin biopsy samples of MF-patients by TCR gamma-PCR, followed by temperature gradient gel electrophoresis (TGGE). In 188 out of 208 MF patients, at least one skin sample with sufficient DNA quality for PCR analysis was obtained. Applying a consensus PCR for the TCR gamma genes V gamma I and J gamma 1/2, we detected monoclonal T cells in 122 cases (65%). In the remaining 66 cases, we performed two multiplex-PCRs, covering rearrangements of the other TCR gamma genes. Here we found in 11 cases (6%) predominant clonal rearrangements of V gamma II-IV and J gamma 1/2 and in 2 (1%) those of V gamma I-IV and J gamma P 1/2. In patients with MF, detecting rearrangements of only V gamma I and J gamma 1/2 is sufficient for PCR screening analysis. In 53 of the patients (28%) the applied methods revealed no monoclonal T cells. This may be due to a low cell number, oligoclonal nature, chromosomal abberations or remaining of TCR in germline configuration.

An approach to the sensitivity of temperature-gradient gel electrophoresis in the detection of clonally expanded T-cells in cutaneous T-cell lymphoma.

Detection of T-cell clonality by polymerase chain reaction (PCR) and high-resolution electrophoresis facilitates differentiation of early stages of cutaneous T-cell lymphoma (CTCL) from benign T-cell-rich dermatoses. However, data regarding the sensitivity of the various electrophoresis techniques differ remarkably. In the present study, the capacity of heteroduplex (HD)-loaded temperature-gradient gel electrophoresis (TGGE) to detect clonally expanded T-cells was assessed systematically and modifications to the procedure were defined. Using our standard protocol, HD-TGGE detected clonal T-cell receptor (TCR)-gamma PCR products, generated from the Jurkat cell line, down to a total of 2 ng/microL (14 ng) DNA. However, slowly migrating single strands of the clonal PCR product reduced the amount of the clonality indicating homoduplices. To overcome this single-strand formation, thus decreasing the detection limit, the urea concentration in the gel and the temperature ramp for the HD-formation were altered, as well as the temperature gradient in the gel. Application of the modified protocol resulted in a tenfold lower detection limit of 0.15 ng/microL (1.05 ng) DNA in the clonal band. The sensitivity of the adapted HD-TGGE was investigated by dilution experiments using the well established T-cell lines Jurkat, Molt-4, MyLa and SeAx. By these approaches clonal PCR products diluted in nonclonal PCR products were detectable down to concentrations of 5-10%. Comparably, in the case of mixtures of clonal in nonclonal DNA the detection limit reached 5-10% clonal DNA. However, by dilution of clonal cells in nonclonal peripheral blood mononuclear cells, which corresponds to in vivo conditions, a lower detection limit of approximately 1-5% was observed.