PEGylation enhances the therapeutic potential for insulin-like growth factor I in central nervous system disorders.

OBJECTIVE: Due to its potent neurotrophic activity, insulin-like growth factor I (IGF-I) has been proposed many times for therapeutic application in disorders of the central nervous system (CNS). However, insufficient brain delivery to yield beneficial central without peripheral side effects have prevented clinical development in most instances. DESIGN: We recently reported the generation of a polyethylene-glycol modified IGF-I variant (PEG-IGF-I) with prolonged half-life and less acute side effects, but with fully maintained slow anabolic activity. Here we investigated if these beneficial properties result in improved brain availability of the drug, thereby reaching therapeutically relevant steady-state concentrations to elicit beneficial effects on neuronal function. RESULTS: After a single subcutaneous injection, PEG-IGF-I reached much higher steady-state levels in brain tissue and cerebrospinal fluid compared with IGF-I. Two weeks treatment with PEG-IGF-I was sufficient to modulate brain plasticity processes, as judged by changes in synaptic proteins and related animal behavior. Furthermore, chronic treatment of a mouse model of brain amyloidosis with PEG-IGF-I reverted deficits in insulin/IGF-I signaling, synaptic proteins and cognitive performance. CONCLUSIONS: Our data generate the therapeutic potential for PEG-IGF-I to treat CNS disorders by systemic drug application, and in addition scientifically support its application in disorders of synaptic function and neuronal development.

Scapuloperoneal syndrome type Kaeser and a wide phenotypic spectrum of adult-onset, dominant myopathies are associated with the desmin mutation R350P.

In 1965, an adult-onset, autosomal dominant disorder with a peculiar scapuloperoneal distribution of weakness and atrophy was described in a large, multi-generation kindred and named 'scapuloperoneal syndrome type Kaeser' (OMIM #181400). By genetic analysis of the original kindred, we discovered a heterozygous missense mutation of the desmin gene (R350P) cosegregating with the disorder. Moreover, we detected DES R350P in four unrelated German families allowing for genotype-phenotype correlations in a total of 15 patients carrying the same mutation. Large clinical variability was recognized, even within the same family, ranging from scapuloperoneal (n = 2, 12%), limb girdle (n = 10, 60%) and distal phenotypes (n = 3, 18%) with variable cardiac (n = 7, 41%) or respiratory involvement (n = 7, 41%). Facial weakness, dysphagia and gynaecomastia were frequent additional symptoms. Overall and within each family, affected men seemingly bear a higher risk of sudden, cardiac death as compared to affected women. Moreover, histological and immunohistochemical examination of muscle biopsy specimens revealed a wide spectrum of findings ranging from near normal or unspecific pathology to typical, myofibrillar changes with accumulation of desmin. This study reveals that the clinical and pathological variability generally observed in desminopathies may not be attributed to the nature of the DES mutation alone, but may be influenced by additional genetic and epigenetic factors such as gender. In addition, mutations of the desmin gene should be considered early in the diagnostic work-up of any adult-onset, dominant myopathy, even if specific myofibrillar pathology is absent.

Gene expression in IFN-gamma-activated murine macrophages

Macrophages are critical for natural immunity and play a central role in specific acquired immunity. The IFN-gamma activation of macrophages derived from A/J or BALB/c mice yielded two different patterns of antiviral state in murine hepatitis virus 3 infection, which were related to a down-regulation of the main virus receptor. Using cDNA hybridization to evaluate mRNA accumulation in the cells, we were able to identify several genes that are differently up- or down-regulated by IFN-gamma in A/J (267 and 266 genes, respectively, up- and down-regulated) or BALB/c (297 and 58 genes, respectively, up- and down-regulated) mouse macrophages. Macrophages from mice with different genetic backgrounds behave differently at the molecular level and comparison of the patterns of non-activated and IFN-gamma-activated A/J or BALB/c mouse macrophages revealed, for instance, an up-regulation and a down-regulation of genes coding for biological functions such as enzymatic reactions, nucleic acid synthesis and transport, protein synthesis, transport and metabolism, cytoskeleton arrangement and extracellular matrix, phagocytosis, resistance and susceptibility to infection and tumors, inflammation, and cell differentiation or activation. The present data are reported in order to facilitate future correlation of proteomic/transcriptomic findings as well as of results obtained from a classical approach for the understanding of biological phenomena. The possible implication of the role of some of the gene products relevant to macrophage biology can now be further scrutinized. In this respect, a down-regulation of the main murine hepatitis virus 3 receptor gene was detected only in IFN-gamma-activated macrophages of resistant mice.

Gene expression in IFN-gamma-activated murine macrophages.

Macrophages are critical for natural immunity and play a central role in specific acquired immunity. The IFN-gamma activation of macrophages derived from A/J or BALB/c mice yielded two different patterns of antiviral state in murine hepatitis virus 3 infection, which were related to a down-regulation of the main virus receptor. Using cDNA hybridization to evaluate mRNA accumulation in the cells, we were able to identify several genes that are differently up- or down-regulated by IFN-gamma in A/J (267 and 266 genes, respectively, up- and down-regulated) or BALB/c (297 and 58 genes, respectively, up- and down-regulated) mouse macrophages. Macrophages from mice with different genetic backgrounds behave differently at the molecular level and comparison of the patterns of non-activated and IFN-gamma-activated A/J or BALB/c mouse macrophages revealed, for instance, an up-regulation and a down-regulation of genes coding for biological functions such as enzymatic reactions, nucleic acid synthesis and transport, protein synthesis, transport and metabolism, cytoskeleton arrangement and extracellular matrix, phagocytosis, resistance and susceptibility to infection and tumors, inflammation, and cell differentiation or activation. The present data are reported in order to facilitate future correlation of proteomic/transcriptomic findings as well as of results obtained from a classical approach for the understanding of biological phenomena. The possible implication of the role of some of the gene products relevant to macrophage biology can now be further scrutinized. In this respect, a down-regulation of the main murine hepatitis virus 3 receptor gene was detected only in IFN-gamma-activated macrophages of resistant mice.

Proteomic analysis of rare molecular species of translated polypeptides from a mouse fetal thymus cDNA library.

Proteomic patterns from an ordered cDNA library of mouse fetal thymus origin of an overall complexity of 1536 clones are described. Patterns have been analyzed at 96, 12, or 8 clones in a mixture, or as individual clones. Clones yield in some instances a single spot, in other cases a complex cluster or family of spots is formed. The determination of the clonal address (a six-digit number, indicating the interception of three pooling dimensions) by inspection of pools of 8, 12 or 16 clones is a reliable approach; nevertheless a complete proof consists in retrieving the clone and then submitting to transcription, translation and proteomic analysis. The spot clusters are meaningful clone identifiers; cluster components (families of polypeptides) are characteristic of individual clones and are independent of clones coexisting (and being co-expressed) in a given pool. A 'cluster' originates from a single cloned message and might be due to post-translational modification (offered by the reticuleocyte machinery) or as a result of programmed degradation. Thirteen clones or families of clonal products are shown, and the heterogeneity of the 'appearance' of clones is documented. In about half, the assignment of a clonal polypeptide product to a storage well position has failed; this might be due to a variety of considerations elaborated herein. The arguments are presented, that analysis of abundant proteins of a cell (activated lymphocyte) comprises 'classical proteomics', but for the analysis of the rare molecular species of proteins, Poissonian approaches of replicable material have to be used.

Global analysis of gene expression in cells of the immune system I. Analytical limitations in obtaining sequence information on polypeptides in two-dimensional gel spots.

We have outlined various aspects and limitations of the collective analysis of protein species of a cell (lymphocyte). We have indicated research directions that, in to our opinion, deserve more attention. We have evaluated mainly the approach used in our laboratory and we recognize that a bulk of important research on the interface of proteomics and genomics remains to be dealt with. It is of great value that we can proceed in our quest by trial and error. But as much as the human genome initiative was not implemented by trial and error, but by formulating new technological approaches, we hope that our approach can be incorporated in the mainstream of proteomics. We need several integrating research directions, some of which are outlined in this communication, namely the use of ordered cDNA libraries, cell-free expression systems, high density filter hybridization, identification of two-dimensional (2-D) gel spots in terms of their amino acid composition through biosynthetic labeling and identification of restriction sites in the corresponding coding sequences. In the accompanying paper the cDNA ordered library approach will be described in some detail.

Increased protein synthesis after T cell activation in presence of cyclosporin A.

BACKGROUND: The immunosuppressive drug, cyclosporin A (CsA), blocks immune responses by inhibiting the calcineurin-dependent dephosphorylation of the nuclear factor of activated T cells (NFAT). We have previously reported that T cells activated in presence of CsA exhibit particular properties. In our study, we have tested the hypothesis that T cells activated in presence of CsA display a differential pattern of gene expression. METHODS: T lymphocytes were activated in vitro by Concanavalin A with or without CsA. The cells were: (1) pulsed with 35S-methionine to label the newly synthesized proteins that in turn were revealed by 2D-gel electrophoresis; (2) analyzed by flow cytometry for activation markers expression; and (3) examined by gel electrophoresis for early tyrosine phosphorylation events. RESULTS: The proteomic patterns of T lymphocytes activated by Concanavalin A, with or without CsA, were compared. In keeping with the well-known effect of the immunosuppressor, many polypeptides were not found in its presence. Remarkably, several newly synthesized polypeptides were detected only when activation was carried out in presence of CsA. In addition, immunologically relevant proteins, such as CD44 and CD69, escape CsA-inhibitory action. Furthermore, CsA did not modify the early protein tyrosine phosphorylation events resulting from T cell triggering. CONCLUSIONS: The present data show that the effect of CsA on protein synthesis is more complex than anticipated. Signaling provided by T cell activation and the blockade of the calcineurin-dependent pathway by CsA results in an altered program of gene expression.

Proteomic analysis of T cell activation in the presence of cyclosporin A: immunosuppressor and activator removal induces de novo protein synthesis.

Cyclosporin A (CsA), a fungal metabolite used in organ transplantation, blocks the immune responses by interfering with early activation signals preventing the induction of the IL2 gene. We have previously reported that the removal of the immunosuppressor provokes the transcription of the IL2 encoding gene. We have now investigated whether the transcription and translation of other genes accompanies this process. Withdrawal of CsA and Concanavalin A (ConA) from cultures of murine T cells activated by ConA in the presence of CsA leads to substantial changes in the pattern of radio-labelled proteins. A large number of polypeptides were synthesised de novo. In addition, a set of polypeptides detected prior to immunosuppressor elimination was not anymore synthesised. Finally, besides these qualitative changes, quantitative differences in terms of increased or decreased polypeptide abundance were also observed. The results demonstrate that activation in the presence of CsA has programmed the T cells to transcribe and translate a large number of genes, without further reactivation, once the immunosuppressor and the activator were removed.

Analysis of a randomly assorted cDNA library from BW 5147 lymphoid cells. Frequency estimates based on computer aided concatenation matching of 2D gel polypeptide products.

A cDNA library was divided into 291 sectors of low complexity, 800-1000 recombinant phage plaques per sector. Aliquots of DNA from sectors prepared from high titer phage were subjected to in vitro transcription using T7 polymerase and thereafter RNA was translated in a cell-free rabbit reticulocyte system in the presence of [35S] methionine. The resulting polypeptides were separated by 2D gel electrophoresis. Radiofluorographs of the gels prepared from 10 sectors were subjected to scanning and image analysis. A multilevel spot matching procedure was employed allowing us to recognise spot occurrence in the 10 sectors. The number of detected polypeptide spots per sector varied between 147 and 325. Overall, 1082 different spots were counted totalling 1983 spots considering multiple entries. The distribution of the spots and the frequency distribution of the RNA population among the 10 sectors are shown. The highest detected frequency is 10(-2), while one half of the RNA molecular species are present at a frequency of 10(-3) or lower.

Neutrophils as a source of putative restriction proteases: degradation of mammalian and yeast proteins monitored by two-dimensional gel electrophoresis.

We have compared the ability of intact neutrophils to degrade a complex substrate of proteins from mammalian and yeast origin. The substrate was obtained by biosynthetic labeling, and subsequent lysis of K562 cells (leukemic cell line) and of yeast culture. The mammalian substrate consisted of 619 and the yeast substrate of 185 different polypeptides, as visualized and represented on two-dimensional gel patterns. Upon incubation of the mammalian substrate with neutrophils, the bulk of spots disappeared so rapidly that after 240 min of incubation only 21 spots were detectable. Just one spot remained unaltered in its intensity throughout the whole period of incubation. About 440 spots reveal a t1/2 shorter than 8 min. Yeast substrate is represented by a smaller number of the starting polypeptides (185) from which 55 spots "survive" the neutrophil treatment. About 30 spots have a t1/2 shorter than 8 min. We conclude that neutrophils are equipped with a potent proteolytic apparatus, and this is capable of eliminating various proteins in a highly efficient manner. The system is much less effective in eliminating proteins from distant species, like yeast. Although the cells governing and regulating the immune system are clearly of lymphoid origin, it might well be that the preimmune task of eliminating self antigens in a manner as predicted in the restriction protease hypothesis is performed by neutrophils.

Restriction sites as identification tags for lymphocyte cDNAs.

A cDNA library was prepared from BW 5147 murine lymphoma cells in lambda ecc III phage and randomly partitioned into 291 sectors, each with 800-1000 recombinant phage plaques. One sector was chosen for further characterization in terms of sensitivity to restriction endonuclease cutting. Aliquots of DNA preparations from this sector were treated with XhoI, SmaI, NcoI, PvuII, PstI, HindIII, EcoRI, BamHI, and ApaLI before being used as templates in a cell-free expression system. The polypeptide products were separated by two-dimensional (2-D) gel electrophoresis and radiofluorographs of the gels were submitted to computer-aided image analysis. The matched patterns were inspected for the presence or absence of spots upon individual endonuclease treatments. Thereafter the results were integrated in a data matrix which served as a basis to construct "restriction tags" for all spots. These (restriction) tags are binary numbers termed "cut numbers" and are a representation of the set of recognition sequences which are (or are not) part of the coding sequence. From 493 sequences (visualized as 2-D gel spots), 12 were not cut by any of the nine enzymes, while 45 were cut by all of them. The percentages of sequences resistant to enzyme treatment ranged between 17% and 77% for NcoI and XhoI, respectively. The enzyme treatments led to the appearance of a certain portion of "new spots", probably products from truncated sequences. From 512 possible cut numbers, 136 were assigned to the 493 spots. Restriction tags are available to facilitate retrieval of cDNA clones from the (partitioned) cDNA library.

Protein expression during murine thymus differentiation.

Driven by our long-standing interest in identifying proteins of the immune system and in characterizing processes involved in lymphocyte differentiation, we studied protein expression in biosynthetically labeled fetal and newborn thymus by 2D gel electrophoresis. Autoradiographs of the gels were scanned with a densitometer and image analysis was performed using the Kepler system. Calibrated polypeptide spot abundances (volumes) were compared to assesses qualitative and quantitative changes of the spot volumes. Among over 300 proteins evaluated at GD (gestation day) 13, 15, and 17, there were sets of proteins that increased and other that decreased in intensity. We could in addition recognize proteins that were completely absent at GD 13 and/or 15 and that appeared thereafter to gradually increase in intensity. Conversely, various polypeptide spots present at early stages (at GD 13 and 15) disappear later (at GD17 or at birth). Among the proteins that increase in intensity prevail molecules with masses less than 35 kD, whereas a considerable portion of those that decrease in intensity are characterized by masses above 60 kD. Spots reported in this communication were not defined beyond tagging them with numbers, which is a prerequisite to follow them up in the proteinpaedia developed in our laboratory. The next step will be to retrieve the coding sequences from the existing partitioned cDNA library (BW 5147) as well as from thymocyte subtraction libraries. We predict that among those polypeptides with varying intensity, important regulatory proteins in thymus development will be found.

Human lymphocyte cDNA ordered library analyzed by 2D gel electrophoresis. 1. Pooling strategy and matching of gel patterns.

We have analyzed an ordered library of 4,608 cDNA clones from the CEM human leukemic cell line. The aim was to facilitate gene retrieval, to enable immediate access to cDNA clones and to provide information on the protein expression of the individual clones in a 2D gel readout. The matrix array of 24 x 16 x 12, each position of which contained lambda jacII phage from one plaque, enabled us to establish pools of clones along the three axes (24 pools of complexity 192 cloned entities, 16 pools of complexity 288 and 12 pools of complexity 384). The total cDNA complexity is here reduced to such a level that spots which in more complex gels served as landmark spots are not present in each pool, and thus cannot serve as landmarks anymore. The image analysis of such gels and especially the matching of spots is not reliable under these circumstances. In order to achieve reliable matching, additional samples were created, such that pools were co-electrophoresed according to a special concatenation scheme; these samples then contained over-lapping elements (e.g., pools 1 + 2 + 3 and 3 + 4 + 5 have at least those spots in common, which originate from pool 3). This approach turned out to be feasible and we have completed the matching of one half of the ordered library. Already from the present stage of analysis we have obtained valuable information on the cDNA library and on the distribution of clones in this library.

Human lymphocyte cDNA ordered library analyzed by 2D gel electrophoresis. 2. Frequency distribution of mRNA populations.

Cell-free transcription and translation products from an ordered library of cDNA clones from the CEM human leukemic cell line were submitted to analysis using two-dimensional gel electrophoresis as a read out system. The matrix array of 24 x 16 x 12 wells contained in each of the positions lambda jacII phages from one plaque. Pools of clones along the three axes (24 x-pools, 16 y-pools and 12 z-pools) were established. Results obtained upon matching of 12 x-pools were scrutinized for estimating the frequency of cDNA molecular species in the library. The results obtained are interpreted in such a way that there are no discrete distributions of mRNA molecular species, but rather there is a continuous distribution of mRNA's covering a wide range of frequencies. The lowest frequency found was about 4.5 x 10(-4) and the highest 1.6 x 10(-2). About half of all clones can be found among these low frequency ones (each occurring 0.45 times among 1,000 clones).

The amino acid composition of 350 lymphocyte proteins.

We determined the amino acid composition of proteins of Sp2 hybridoma cells by a procedure which assembles the information on the polypeptides upon two-dimensional gel electrophoresis, such that biosynthetic labeling with 20 different 3H amino acids provides the data--spot intensities--on the relative representation of the detected polypeptides. The gels were impregnated with 2,5-diphenyloxazol (PPO) and suitably exposed radiofluorographs were selected for analysis. The images originating from the 12 cultures labeled with amino acids R, A, H, I, L, K, M, F, P, S, T and Y were analysed with the Kepler image analysis system. The spot volume data of the 12 analysed patterns were corrected for the unequal labeling efficiencies of the 3H amino acids and for the various exposure times. This correction is performed by applying calibration factors based on the amino acid determination of a hydrolysate of the analysed cells. After the calibration step was applied to the data files we used the amino acid compositions of nine proteins taken from a database to establish for each of these proteins the correlation coefficients with the image analysis derived amino acid compositions of all spots. The correlation coefficients allow us to tentatively identify polypeptide spots on two-dimensional gels, while the amino acid composition of 350 investigated two-dimensional gel spots is usable as an identification tag in the gene retrieval from our cDNA libraries.

Thymus-grafted SCID mice show transient thymopoiesis and limited depletion of V beta 11+ T cells.

To seek direct evidence for the notion that stem cells in the thymus need to be constantly replenished from the bone marrow (BM), fetal (day 15) thymuses from normal BALB/c mice were grafted into T and B cell-deficient C.B-17 SCID mice (both H-2d, I-E+). The thymus grafts in these mice showed normal thymopoiesis for the first 3 wk postgrafting but then developed sudden atrophy with near complete loss of CD4+8+ cells by 4-5 wk. Such atrophy was not seen when the thymus-grafted mice were cotransplanted with normal BM cells. The lymph nodes of SCID mice receiving thymus grafts alone contained mature T cells but virtually no B cells. This lack of B cells was associated with aberrant I-E-restricted V beta deletion: the depletion of V beta 3+ and V beta 5+ T cells was near complete, whereas V beta 11+ cells showed only marginal depletion.

Inhibition of proliferation by retinoids, cytokines and their combination in four human transformed epithelial cell lines.

Various combinations of retinoids and cytokines were examined for their synergistic effect on inhibition in proliferation of four human transformed epithelial cell lines, MCF7 (mammary carcinoma), SCC4, SCC15 and A431 (squamous cell carcinomas). Synergism depended on the cell line tested, to some degree on the specific retinoid but particularly on the type of cytokine used. IFN alpha had the widest spectrum of activity. IFN gamma, TNF-alpha, IL-1, EGF and TGF-beta also exerted a synergistic effect on proliferation inhibition in certain cell lines, whereas G-CSF was inactive. Association of retinoids and cytokines represents a new approach to antitumor chemotherapy.

Antiproliferative activity of retinoids, interferon alpha and their combination in five human transformed cell lines.

Tretinoin, isotretinoin and acitretin were examined for their capacity to modulate the proliferation of the cell lines: HL-60 (acute promyelocytic leukemia), MCF7 (mammary carcinoma), SCC4, SCC15 and A431 (squamous cell carcinomas). Retinoids inhibited proliferation to a varying extent in all 5 cell lines. The cytokine IFN alpha had a significant antiproliferative effect only on HL-60, SCC4 and SCC15. The combination of retinoids with IFN alpha led in all 5 cell lines to a more profound reduction in proliferation than either retinoids or IFN alpha alone.

Efficient purification of recombinant human tumor necrosis factor beta from Escherichia coli yields biologically active protein with a trimeric structure that binds to both tumor necrosis factor receptors.

A fast and efficient method for medium scale purification of recombinant human tumor necrosis factor beta (rTNF-beta) from Escherichia coli cells is described. The purified rTNF-beta displayed biological activity similar to rTNF-alpha in a WEHI 164 cell cytotoxicity assay. The titration curve of rTNF-beta and elution profiles of rTNF-beta in gel filtration experiments were different from those of rTNF-alpha. However, light scattering and ultra-centrifugation studies showed that both cytokines have trimeric structures in solution at 0.5 mg/ml, with minor differences in the distribution of nontrimeric species. rTNF-beta bound to purified 55- and 75-kDa TNF receptors with high affinity. The binding of rTNF-beta to either receptor was analyzed on Scatchard plots and compared with that of rTNF-alpha.

The principal tumor necrosis factor receptor in monocyte cytotoxicity is on the effector cell, not on the target cell.

Several tumor target cell lines, prototypically K562 cells, are resistant to lysis by recombinant tumor necrosis factor (TNF alpha) but are killed by monocytes expressing membrane-associated TNF, suggesting that membrane TNF could account for monocyte-mediated cytotoxicity. Formaldehyde-fixed monocytes or extracted monocyte membrane fragments are cytotoxic to K562 target cells. Treatment of monocytes with interferon-gamma (IFN-gamma) increases cytotoxicity by live and fixed cells or by extracted monocyte membranes. Both TNF and TNF receptors are detectable on monocyte membranes by FACS analysis, and the levels of each are modulated by treatment with IFN-gamma. Cytotoxicity can be inhibited by either anti-TNF or anti-TNF receptor antibodies. Incubation of effector cells with exogenous soluble TNF prior to fixation or membrane preparation increases their cytotoxicity. In contrast, incubation of the target cells with exogenous TNF neither increases nor decreases killing by effector cell membrane fragments or intact effector cells. The data suggest that the TNF receptors on the effector cell, but not on the target cell, play a crucial role in TNF-mediated cytotoxicity.