[Periodic fever and pancytopenia in a 35-year-old patient]. / Periodisches Fieber und Panzytopenie bei einem 35­jährigen Patienten.

MEDICAL HISTORY AND INITIAL PRESENTATION: A 35-year-old patient with a previous history of persistent episodic fever, sore throat, myalgia, and cephalgia presented for evaluation of pancytopenia. He had no recent travel history, except for a stay in Italy 1 year prior to admission and in Spain several years in the past. DIAGNOSTIC WORKUP: Laboratory evaluation confirmed pancytopenia, agranulocytosis, and elevated infection parameters without indicative serological results en par with lymphadenitis colli. Computed tomography scanning revealed cervical lymphadenopathy, hepatosplenomegaly, and colitis with occult perforation of the sigmoid colon. Bone marrow biopsy showed an infiltration of polyclonal plasma cells. Lymph node biopsy was compatible with necrotizing lymphadenitis. DIAGNOSIS: Polymerase chain reaction analysis of a lymph node specimen confirmed the presence of Leishmania species, thereby enabling the diagnosis of visceral Leishmania. THERAPY COURSE: Treatment with liposomal amphotericin B was initiated. Both fever and lymphadenopathy quickly resolved. CONCLUSION: VL is a clinically pleiotropic, severe disease with fatal outcome if left untreated. It often presents with distinct similarities to hematologic malignancies. Exacerbation can occasionally occur as fulminant macrophage activation syndrome. Disease incidence is globally increasing and has not peaked as yet. A complex interplay between pathogen and the immune system is the key pathophysiological mechanism.

Optimization of design and production strategies for novel adeno-associated viral display peptide libraries.

Libraries displaying random peptides on the surface of adeno-associated virus (AAV) are powerful tools for the generation of target-specific gene therapy vectors. However, for unknown reasons the success rate of AAV library screenings is variable and the influence of the production procedure has not been thoroughly evaluated. During library screenings, the capsid variants with the most favorable tropism are enriched over several selection rounds on a target of choice and identified by subsequent sequencing of the encapsidated viral genomes encoding the library capsids with targeting peptide insertions. Thus, a high capsid-genome correlation is crucial to obtain the correct information about the selected capsid variants. Producing AAV libraries by a two-step protocol with pseudotyped library transfer shuttles has been proposed as one way to ensure such a correlation. Here we show that AAV2 libraries produced by such a protocol via transfer shuttles display an unexpected additional bias in the amino-acid composition which confers increased heparin affinity and thus similarity to wildtype AAV2 tropism. This bias may fundamentally impair the intended use of AAV libraries, discouraging the use of transfer shuttles for the production of AAV libraries in the future.

Treatment of multifocal breast cancer by systemic delivery of dual-targeted adeno-associated viral vectors.

Adeno-associated viral (AAV) vectors yield high potential for clinical gene therapy but, like for other vectors systems, they frequently do not sufficiently transduce the target tissue and their unspecific tropism prevents their application for multifocal diseases such as disseminated cancer. Targeted AAV vectors have been obtained from random AAV display peptide libraries but so far, all vector variants selected from AAV libraries upon systemic administration in vivo retained some collateral tropism, frequently the heart. Here we explored, if this impediment can be overcome by microRNA-regulated transgene cassettes as the combination of library-derived capsid targeting and micro-RNA control has not been evaluated so far. We used a tumor-targeted AAV capsid variant (ESGLSQS) selected from random AAV-display peptide libraries in vivo with remaining off-target tropism toward the heart and regulated targeted transgene expression in vivo by complementary target elements for heart-specific microRNA (miRT-1d). Although this vector still maintained its strong transduction capacity for tumor target tissue after intravenous injection, transgene expression in the heart was almost completely abrogated. This strong and completely tumor-specific transgene expression was used for therapeutic gene transfer in an aggressive multifocal, transgenic, polyoma middle T-induced, murine breast cancer model. A therapeutic suicide gene, delivered systemically by this dual-targeted AAV vector to multifocal breast cancer, significantly inhibited tumor growth after one single vector administration while avoiding side effects compared with untargeted vectors.

Novel random peptide libraries displayed on AAV serotype 9 for selection of endothelial cell-directed gene transfer vectors.

We have demonstrated the potential of random peptide libraries displayed on adeno-associated virus (AAV)2 to select for AAV2 vectors with improved efficiency for cell type-directed gene transfer. AAV9, however, may have advantages over AAV2 because of a lower prevalence of neutralizing antibodies in humans and more efficient gene transfer in vivo. Here we provide evidence that random peptide libraries can be displayed on AAV9 and can be utilized to select for AAV9 capsids redirected to the cell type of interest. We generated an AAV9 peptide display library, which ensures that the displayed peptides correspond to the packaged genomes and performed four consecutive selection rounds on human coronary artery endothelial cells in vitro. This screening yielded AAV9 library capsids with distinct peptide motifs enabling up to 40-fold improved transduction efficiencies compared with wild-type (wt) AAV9 vectors. Incorporating sequences selected from AAV9 libraries into AAV2 capsids could not increase transduction as efficiently as in the AAV9 context. To analyze the potential on endothelial cells in the intact natural vascular context, human umbilical veins were incubated with the selected AAV in situ and endothelial cells were isolated. Fluorescence-activated cell sorting analysis revealed a 200-fold improved transduction efficiency compared with wt AAV9 vectors. Furthermore, AAV9 vectors with targeting sequences selected from AAV9 libraries revealed an increased transduction efficiency in the presence of human intravenous immunoglobulins, suggesting a reduced immunogenicity. We conclude that our novel AAV9 peptide library is functional and can be used to select for vectors for future preclinical and clinical gene transfer applications.

Predictive preoperative clinical score for patients with liver-only oligometastatic colorectal cancer.

BACKGROUND: Resection of liver metastases from colorectal cancer (CRC) in the oligometastatic stage improves survival and is a potentially curative treatment. Thus, predictive scores that reliably identify those patients who especially benefit from surgery are essential. PATIENTS AND METHODS: In this multicenter analysis, 512 patients had undergone surgery for liver metastases from CRC. We investigated distinct cancer-specific risk factors that are routinely available in clinical practice and developed a predictive preoperative score using a training cohort (TC), which was thereafter tested in a validation cohort (VC). RESULTS: Inflammatory response to the tumor, a right-sided primary tumor, multiple liver metastases, and node-positive primary tumor were significant adverse variables for overall survival (OS). Patients were stratified in five groups according to the cumulative score given by the presence of these risk factors. Median OS for patients without risk factors was 133.8 months [95% confidence interval (CI) 81.2-not reached (nr)] in the TC and was not reached in the VC. OS decreased significantly for each subsequent group with increasing number of risk factors. Median OS was significantly shorter (P < 0.0001) for patients presenting all four risk factors: 14.3 months (95% CI 10.5 months-nr) in the TC and 16.6 months (95% CI 14.6 months-nr) in the VC. CONCLUSIONS: Including easily obtainable variables, this preoperative score identifies oligometastatic CRC patients with prolonged survival rates that may be cured, and harbors potential to be implemented in daily clinical practice.

Heart-targeted adeno-associated viral vectors selected by in vivo biopanning of a random viral display peptide library.

Selection of targeted vectors from virus display peptide libraries is a versatile and efficient approach to improve vector specificity and efficiency. This strategy has been used to target various cell types in vitro. Here, we report the screening of an adeno-associated virus type 2 (AAV2) display peptide library in vivo to select vectors specifically homing to heart tissue after systemic application in mice. Selected library clones indicated superior specificity of gene transfer compared with wild-type AAV2, AAV9 and a heparin binding-deficient AAV2 mutant. Such targeted vectors were able to reconstitute expression of delta-sarcoglycan in the heart of adult delta-sarcoglycan knockout mice after systemic gene transfer in vivo, attesting to the therapeutic potential of this approach.

Leukemia-targeting ligands isolated from phage-display peptide libraries.

Ligands specifically binding to leukemia cells may be used for drug targeting, resulting in more effective treatment with less side effects. Little is known about receptors specifically expressed on acute myeloid leukemia (AML) cells or ligands thereof. We selected random phage display peptide libraries on Kasumi-1 AML cells. A peptide with the sequence CPLDIDFYC was enriched. Phage displaying this peptide strongly bound to Kasumi-1 and SKNO-1 cells and binding could be inhibited by the cognate peptide. Both, Kasumi-1 and SKNO-1 cells carry the chromosomal translocation t(8;21), leading to aberrant expression of the fusion protein AML1/ETO. CPLDIDFYC also strongly and specifically bound primary AML1/ETO-positive AML blasts as well as U-937 cells with forced AML1/ETO expression, suggesting that the CPLDIDFYC receptor may be upregulated upon AML1/ETO expression. Gene expression profiling comparing a panel of CPLDIDFYC-binding and CPLDIDFYC-nonbinding cell lines identified a set of potential receptors for the CPLDIDFYC peptide. Further analysis suggested that alpha4beta1 integrin (VLA-4) is the CPLDIDFYC receptor. Finally, we showed that the CPLDIDFYC-phage is internalized upon receptor binding, suggesting that the CPLDIDFYC-receptor-ligand interaction may be exploitable for targeting drugs or gene therapy vectors to leukemia cells carrying the suitable receptor.

Modulation of the immune response by systemic targeting of antigens to lymph nodes.

Factors that determine the immunogenicity of an antigen in vivo are still largely unknown. Direct administration of antigens into lymphatic organs appears to enhance immune response. We hypothesized that systemically targeting antigens to lymphatic tissue in vivo might modulate immunity. To test this hypothesis, we measured the humoral immune response elicited by bacteriophage vaccination. We show that the responses against a lymph node-targeted phage are significantly higher than those against control untargeted phage; the effect is specific because it is inhibited by coadministration of the cognate synthetic peptides displayed. Our data suggest that systemic targeting of antigens to lymph nodes through the circulation modulates humoral immune response. This strategy may have broad applications in the development of vaccines, production of antibodies, and immunotherapy.

A new look at the role of p53 in leukemia cell sensitivity to chemotherapy.

A gene encoding the p53 val135 mutant, which assumes mutant conformation at 38.5 degrees C and wild-type conformation at 32.5 degrees C, was introduced into p53-deficient K562 myeloid leukemia cells. Forced expression of wild-type, but not mutant, p53 resulted in growth arrest, accumulation of p21 and Bax proteins, and delayed cell death. Wild-type p53 enhanced the cytotoxic effects of some drugs and attenuated those of others. Compared with wild-type p53, mutant p53 induced much stronger sensitization to drug cytotoxicity. This occurred in the absence of effects on cell cycle progression or activation of several p53 target genes. Although both mutant and wild-type p53 induced changes of immunophenotype, no specific pattern of differentiation was associated with enhanced chemosensitivity. Thus, (1) induction of growth arrest and activation of p53 target genes such as p21 and bax are linked to the wild-type conformation of p53; (2) p53 induces immunophenotypic changes of myeloid leukemia cells suggestive of multidirectional differentiation in a conformation-dependent manner; and (3) (so-called) mutant p53 induces chemosensitization in the absence of effects on cell cycle progression, activation of bax, p21, gadd45 and mdm-2, or a specific pattern of differentiation; and (4) chemosensitization mediated by wild-type p53 may be masked by transcription-dependent induction of growth arrest.

Molecular adaptors for vascular-targeted adenoviral gene delivery.

Gene therapy would be considerably more effective if vectors could be targeted to specific organs or tissues after systemic administration. We previously developed an in vivo selection system to isolate organ- and tumor-homing peptides from phage display peptide libraries. The peptides isolated by this approach bind to receptors expressed in vascular endothelia. We describe here the development of molecular adaptors to target adenoviral gene therapy vectors to selective vascular "addresses." The adaptor design consists of an organhoming peptide conjugated to an adenovirus-binding moiety. We isolated and characterized several monoclonal antibodies that bind to adenovirus type 5 (Ad5). Two of the antibodies neutralized Ad5 infection. We linked the Fab fragments of one of these antibodies to a synthetic lung-homing peptide (CGFECVRQCPERC or GFE-1 peptide) and tested the ability of the resulting bispecific conjugate to retarget Ad5. Cells that express the receptor for the GFE-1 peptide and are resistant to Ad5 infection were sensitized to recombinant Ad5 vectors in the presence of the Fab-GFE adaptor. Our findings indicate that selective gene therapy delivery may be developed on the basis of our vascular targeting technology.

Potentiation of treosulfan toxicity by the glutathione-depleting agent buthionine sulfoximine in human malignant glioma cells: the role of bcl-2.

Median survival of human malignant glioma patients is less than one year even with cytoreductive surgery and postoperative radiotherapy. Adjuvant chemotherapy has been rather ineffective. Here, we studied the potentiation by L-buthionine-[S,R]-sulfoximine (BSO), a glutathione-depleting agent, of anticancer drug actions on two human malignant glioma cell lines, LN-229 and T98G. LN-229 has wild-type p53 status, T98G is mutant for p53. Glutathione levels were depleted by BSO with similar kinetics in both cell lines. Only LN-229 cells were growth-inhibited by BSO. BSO had minor effects on the toxicity of doxorubicin, ACNU (1-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-3-(2-chloroethyl)-3-nitrosou rea, nimustine) and vincristine. BSO failed to alter teniposide or cytarabine toxicity. BSO induced prominent sensitization to the alkylating agent, treosulfan, in both cell lines, as assessed by viability assays, in situ DNA end labeling and quantitative DNA fragmentation. Treosulfan is thought to mediate toxicity via formation of reactive epoxides. In the absence of BSO, treosulfan had little acute cytotoxic and moderate antiproliferative effects. Synergistic glioma cell cytotoxicity induced by treosulfan and BSO was not associated with reactive oxygen species formation. Ectopic expression of bcl-2 did not alter basal glutathione levels but attenuated glutathione depletion induced by BSO. Bcl-2 provided only moderate protection from synergistic induction of glioma cell death by treosulfan and BSO. Glutathione depletion may play a role in BSO-mediated chemosensitization, but other mechanisms are probably involved as well. BSO may be a useful agent for glioma cell sensitization to specific chemotherapeutic drugs such as treosulfan.

The influence of genetic sex on sexual differentiation of diencephalic dopaminergic neurons in vitro and in vivo.

Soma sizes of embryonic male and female tyrosine hydroxylase-immunoreactive neurons were measured in 3 diencephalic regions in situ and in diencephalic dissociated cell cultures. Male neurons were about 30% larger than female neurons both in vitro and in situ. Treatment of cultures with sex steroids did not affect the sex differences. It is concluded that sexual differentiation of dopaminergic neurons may be under primary genetic control.

Hypericin-induced apoptosis of human malignant glioma cells is light-dependent, independent of bcl-2 expression, and does not require wild-type p53.

Hypericin and tamoxifen are experimental agents for the adjuvant chemotherapy of malignant glioma. We report that hypericin and tamoxifen induce apoptosis of 7 human malignant glioma cell lines in a concentration- and time-dependent manner. Illumination is essential for the cytotoxicity of hypericin but not tamoxifen. Apoptosis is unaffected by inhibitors of RNA and protein synthesis or free radical scavengers, does not require wild-type p53 activity, and occurs in glioma cells expressing high levels of bcl-2. There is no correlation between hypericin and tamoxifen-induced cytotoxicity and inhibition of protein kinase C (PKC). Ectopic expression of a murine bcl-2 transgene provides modest protection from tamoxifen but does not affect hypericin toxicity. Hypericin and tamoxifen do not modulate glioma cell killing induced by tumor necrosis factor-alpha (TNF-alpha) or CD95 ligand. Both drugs augment the acute cytotoxicity of various cancer chemotherapy drugs but fail to shift their EC50 values in modified colony formation assays. These data do not provide further supportive evidence how to enhance the limited efficacy of tamoxifen treatment for human malignant glioma. However, hypericin is a promising agent for the treatment of malignant glioma if local photodynamic activation of hypericin in the glioma tissue can be achieved.

Immunochemotherapy of malignant glioma: synergistic activity of CD95 ligand and chemotherapeutics.

Malignant glioma cells are susceptible to CD95(Fas/APO-)-mediated apoptosis triggered by agonistic antibody. Here we examined the proapoptotic effects of the natural CD95 ligand, a cytotoxic cytokine homologous to tumor necrosis factor, on malignant glioma cell lines LN-229, LN-308 and T98G. We assessed whether glioma cell killing is synergistically enhanced by cotreatment with CD95 ligand and chemotherapeutic agents, including doxorubicin, carmustine, vincristine, etoposide, teniposide, 5-fluorouracil and cytarabine. Synergy was examined at low concentrations of cytotoxic drugs and CD95 ligand with a defined effect level (IC15). Short-term-cytotoxicity assays showed prominent killing of the glioma cells by CD95 ligand but not by the drugs at relevant concentrations. CD95 ligand induced apoptosis in the acute toxicity paradigm was augmented by doxorubicin and vincristine. Growth-inhibition assays revealed prominent synergy between CD95 ligand and all drugs examined. The best synergy was obtained with CD95 ligand and doxorubicin, vincristine or teniposide. The strong synergistic antiproliferative effects were observed at much lower concentrations of CD95 ligand and cytotoxic drugs than the moderate synergistic acute cytotoxic effects. All cell lines examined express the Bcl-2 protein. LN-229 has partial wild-type p53 activity. T98G has mutant p53, LN-308 has a deleted p53 gene and lacks p53 protein expression. Thus, synergistic effects of CD95 ligand and cytotoxic drugs were observed in cell lines exhibiting two features thought to play a role in the chemoresistance of human malignant glioma cells: loss of wild-type p53 activity and acquisition of bcl-2 expression. Ectopic expression of murine bcl-2 conferred partial protection from CD95 ligand and drugs when administered alone but did not interfere with the mechanisms underlying the synergistic effects of CD95 ligand and chemotherapeutic drugs.

Chemosensitivity of human malignant glioma: modulation by p53 gene transfer.

Loss of wild-type p53 activity is one of the most common molecular abnormalities in human cancers including malignant gliomas. The p53 status is also thought to modulate sensitivity to irradiation and chemotherapy. Here, we studied the effect of a p53 gene transfer on the chemosensitivity of three human glioma cell lines with different endogenous p53 status (LN-229, wild-type; LN-18, mutant; LN-308, deleted), using the murine temperature-sensitive p53 val135 mutant. Expression of mutant p53 enhanced proliferation of LN-308 cells but reduced proliferation in the other cell lines. Expression of wild-type p53 caused reversible growth arrest of all cell lines but failed to induce apoptosis. Growth arrest induced by wild-type p53 was associated with strong induction of p21 expression. Strong induction of BAX expression and loss of BCL-2 expression, which are associated with p53-dependent apoptosis rather than growth arrest, were not observed. Wild-type p53 failed to sensitize glioma cells to cytotoxic drugs including BCNU, cytarabine, doxorubicin, teniposide and vincristine. The combined effects of wild-type p53 gene transfer and drug treatment were less than additive rather than synergistic, suggesting that the intracellular cascades activated by p53 and chemotherapy are redundant. Unexpectedly, forced expression of mutant p53 modulated drug sensitivity in that it enhanced the toxicity of some drugs but attenuated the effects of others. These effects may represent a dominant negative effect of mutant p53 in LN-229 cells which have wild-type p53 activity but must be considered a gain of function-type effect in the other two cell lines which have no wild-type p53 activity. Importantly, no clear-cut pattern emerged among the three cell lines studied. We conclude that somatic gene therapy based on the reintroduction of p53 will limit the proliferation of human malignant glioma cells but is unlikely to induce clinically relevant sensitization to chemotherapy in these tumors.

Incorporation of tumor-targeting peptides into recombinant adeno-associated virus capsids.

The human parvovirus adeno-associated virus type 2 (AAV-2) possesses many features that make it an attractive vector for gene delivery in vivo. However, its broad host range may limit its usefulness and effectivity in several gene therapy applications in which transgene expression needs to be limited to a specific organ or cell type. In this study, we explored the possibility of directing recombinant AAV-2 transduction by incorporating targeting peptides previously isolated by in vivo phage display. Two putative loops within the AAV-2 capsid were examined as sites for incorporation of peptides. We tested the effects of deleting these loops and different strategies for the incorporation of several targeting peptides. The tumor-targeting sequence NGRAHA and a Myc epitope control were incorporated either as insertions or as replacements of the original capsid sequence. Viruses were assessed for packaging, accessibility of incorporated peptides, heparin binding, and transduction in a range of cell lines. Whereas recombinant viruses containing mutant capsid proteins were produced efficiently, transduction of several cell lines was significantly impaired for most modifications. However, certain mutants containing the peptide motif NGR, which binds CD13 (a receptor expressed in angiogenic vasculature and in many tumor cell lines), displayed an altered tropism toward cells expressing this receptor. Based on this work and previous studies, possible strategies for achieving in vivo targeting of recombinant AAV-2 are discussed.