High-efficiency transformation of Saccharomyces cerevisiae cells by bacterial minicell protoplast fusion.

After a new transformation procedure, 10% of Saccharomyces cerevisiae cells were found to contain transforming DNA sequences. We used direct transfer of plasmid molecules by fusing bacterial minicell protoplasts to yeast protoplasts. Since the procedure significantly reduces the toxic effect of procaryotic protoplasm on the eucaryotic organism, it might be generally applicable in other systems in which transformation is inefficient or impossible.

Budesonide, but not tacrolimus, affects the immune functions of normal human keratinocytes.

Topical immunosuppressant therapy is widely used in the treatment of inflammatory skin diseases such as psoriasis and atopic dermatitis. Besides its beneficial therapeutic effects, application of topical anti-inflammatory drugs may render the epidermis more vulnerable to invading pathogens by suppressing innate immune responses in keratinocytes, such as cytokine production and Toll-like receptor (TLR) expression. In order to evaluate and compare the immunosuppressive effects of different immunosuppressant drugs on keratinocytes, we treated lipopolysaccharide (LPS)-stimulated and -unstimulated normal human keratinocytes with the synthetic corticosteroid budesonide and the macrolide tacrolimus. The expressions of the pattern recognition receptors (PRRs) TLR2 and TLR4 were measured by quantitative RT-PCR, pro-inflammatory cytokines IL-1alpha, IL-8 and TNF-alpha were monitored by quantitative RT-PCR and by ELISA, and alterations in TLR2 protein level were measured by flow cytometry. Budesonide had a suppressive effect on both constitutive and LPS-induced IL-8 gene expression. The amount of TNF-alpha mRNA was diminished in unstimulated keratinocytes, while TLR2 mRNA expression was markedly enhanced both in unstimulated and LPS-treated cells after incubation with budesonide. This increase in TLR2 mRNA expression was also detectable at the protein level in LPS-stimulated cells. Tacrolimus had no effect on any of the examined genes. Budesonide, but not tacrolimus, significantly inhibited the NF-kappaB-dependent luciferase reporter activity in HaCaT cells after induction with LPS or TNF-alpha. Although tacrolimus and budesonide are both effective treatments in some inflammatory skin diseases, the data provided here imply differences in local therapeutic and adverse effects of these two topical immunosuppressants.

Mycosis fungoides involving the central nervous system.

The clinical records of nine patients, followed at the University of Chicago, with mycosis fungoides (MF) and neurologic deficits due to direct CNS involvement were reviewed. Lymph node and visceral involvement was present in all patients. Clinical findings included cranial nerve deficits in four patients, altered mental status in eight, gait disturbances in three, and leg weakness in one. Helpful diagnostic studies included lumbar puncture with CSF cytology, computerized tomography (CT) brain scans, brain biopsy, and myelogram. Two patients, who were treated with radiation therapy (RT), survived 14 and 40 weeks, respectively. Survival of less than 4 weeks limited treatment in the other patients. Autopsies were performed on six patients and revealed meningeal infiltration and perivascular infiltration of the brain. In addition, the cerebral parenchyma was infiltrated in one patient and a lymphomatous tumor was found in the brainstem of another. We conclude that the varied clinical and pathologic features of CNS involvement with mycosis fungoides resemble those seen in leukemias and other lymphoproliferative disorders. Better awareness of the clinical features may permit earlier diagnosis and initiation of therapy.

Platelet-activating factor antagonists (BN 52021 and BN 50730) inhibit tumor necrosis factor-alfa-mediated cytotoxicity on murine L929 tumor cells.

Tumor necrosis factor (TNF)-alfa has been described as a mononuclear phagocyte-produced cytotoxin that causes the necrosis and regression of some tumors. The mechanism of the cytotoxicity and the basis for the differential cytotoxic effects of TNF against cells of various origin remains unclear. It has also been reported, that murine TNF stimulates the production of platelet-activating factor (PAF) by cultured peritoneal macrophages, and that PAF enhances TNF production by alveolar macrophages. Furthermore, it is known that the synthesis and release of PAF are inhibited by plasma proteinase inhibitors. This study was devoted to investigate the effects of two specific PAF antagonists (BN 52021 and 50730), and a proteinase inhibitor (aprotinin; GordoxR) on the TNF-induced cytotoxicity in L929 murine fibroblasts. Our present findings indicate that TNF-induced cytotoxicity is inhibited in a dose-dependent manner by the PAF antagonists studied and by the kallikrein inhibitor aprotinin. These findings provide further evidence suggesting that PAF might be involved in the process of the TNF-alfa-induced cytotoxicity of L929 mouse fibroblasts.

TNF inhibits myogenesis and downregulates the expression of myogenic regulatory factors myoD and myogenin.

The presence of TNF and other inflammatory cytokines and their receptors is detected during embryonic development, but our knowledge about the role of these proteins in differentiation and development is very limited. TNF modulates the synthesis and activity of a number of transcriptional proteins that regulate the activity of tissue specific genes, therefore it may play a role in normal development. Since its synthesis is upregulated by stress and infections, it may also participate in the induction of pathological developmental processes and malformation. We investigated the effect of TNF in an in vitro differentiation system using C2 myoblasts. This inflammatory cytokine exerted a positive effect on the early steps of the process: it enhanced the proliferation and aggregation of myoblast cells. In contrast, TNF strongly inhibited the expression of those myogenic transcription factors (myoD and myogenin), which are known to be responsible for upregulated activity of muscle specific genes (like the genes of the myofilament proteins), and blocked the synthesis of mRNAs of myogenic differentiation markers (like skeletal alpha-actin, myosin heavy and light chains). As a result, these cells did not synthesize myofilament proteins and the organization of myofilaments did not take place in TNF-treated myoblasts.

45T-1, an established cell line with characteristics of Sertoli cells, forms organized aggregates in vitro after exposure to tumor necrosis factor alpha.

In the testis TNF is produced by germinal cells. The putative role of tumor necrosis factor alpha (TNF) in development and differentiation was investigated in 45T-1 mouse cell cultures, a cell line with characteristic markers of Sertoli cells, established from transgenic mouse families expressing the polyoma large T antigen in their testes. Exposure to TNF elicited a gradual assembly of the cells of the monolayer into highly organized spheroids. The first morphological sign of the changes was detected one week after TNF treatment by anti-desmin immunostaining which showed the formation of foci in the culture consisting of several hundred cells connected by an increasing number of cell contacts. Between days 10-20 the cells formed large ovoid or vermiform aggregates covered by several layers of flat, elongated cells. These cells extended septae into the inner mass of the spheroids consisting of loosely arranged, large polygonal or palisadic cells. The spheroids were surrounded by radially arranged elongated cells covered by small blebs. TNF treatment upregulated laminin expression in 45T-1 cell cultures, which is known to induce formation of cord-like structures by Sertoli cells in vitro. Coculturing 45T-1 cells with immortalized germinal cells or TNF-producing HeLa cells also lead to the formation of spheroids. These observations suggest that TNF production of germinal cells might contribute to the organization/differentiation of Sertoli cells.

Cloning and characterization of the genes of the CeqI restriction-modification system.

Two genes from Corynebacterium equii, a Gram-positive bacterium producing the CeqI restriction-modification enzymes were cloned and sequenced. In vivo restriction experiments, DNA and amino acid sequence data suggest that the two genes code for the endonuclease and the methyltransferase enzymes. However, when the two genes are expressed in E. coli, practically no enzyme activity can be detected in the supernatants of sonicated cells. Based on the DNA sequence data CeqI restriction endonuclease (an EcoRV izoschizomer) consists of 270 amino acid residues with a predicted molecular mass of 31.6 kDa, in good agreement with the previously measured 32 +/- 2 kDa. The methyltransferase is 517 residues long (approx. 60 kDa). The two genes are in opposite orientation and overlap by 37 base pairs on the chromosome. The deduced amino acid sequence of the putative endonuclease gene revealed long stretches of hydrophobic amino acids, that may form the structural basis of the unusual aggregation properties of the restriction endonuclease. The amino acid sequence of the methylase shows homologies with other type II methyltransferases.

Effect of RU 38486 on TNF production and toxicity.

Glucocorticoid steroids provide considerable protection against the systemic toxicity of tumor necrosis factor-alpha (TNF-alpha, cachexin). In animal experiments RU 38486 (mifepristone), a steroid antagonist, increased the synthesis of TNF and sensitized the animals to the cytotoxic action of TNF. As compared to the control and methylprednisolone-treated groups, mifepristone significantly increased the level of TNF in the serum, liver and spleen of lipopolysaccharide (LPS)-treated animals. In tissue cultures RU 38486 induced the TNF synthesis of myeloid cells and increased the TNF production of genetically modified HeLa cells, which synthesize TNF constitutively. Normal and tumor cell cultures exhibited increased sensitivity toward TNF in the presence of mifepristone.

Presynaptic inhibitory effect of TNF-alpha on the release of noradrenaline in isolated median eminence.

The effect of tumor necrosis factor-alpha (TNF-alpha) on the stimulation-evoked release of noradrenaline (NA) from isolated rat median eminence (ME) was investigated, using a low-volume perfusion system. Median eminence, loaded with [3H]noradrenaline, was superfused with Krebs solution and stimulated electrically (2 Hz, 120 shocks). The effect of TNF-alpha was studied on the S2/S1 ratio. It was found that stimulation-evoked release of NA from noradrenergic axon terminals in the isolated rat ME was inhibited by TNF-alpha and this effect was concentration-dependent. In contrast, TNF-alpha had no effect on the release of [3H]NA from the spleen. Since NA released in the ME might be involved in the modulation of corticotropin-releasing factor (CRF) production, it is suggested that TNF-alpha, through presynaptic modulation of NA release from noradrenergic nerve terminals in the ME, might regulate CRF and other neurohormone release in this hypothalamic structure.

Pathophysiological effects of human TNF-alpha-producing tumor xenografts in immunosuppressed mice.

Groups of CBA mice immunosuppressed with anti-thymocyte serum (ATS) treatment were xeno-transplanted with either HeLa human cervical carcinoma cells or genetically modified cells expressing the human tumor necrosis factor-alpha (TNF) gene (All cells). Both cell lines were highly resistant to the cytotoxic effects of TNF. If 3 x 10(6) tumor cells were inoculated s.c. into female mice, HeLa cells grew progressively into large tumors and killed 74% of the recipients, while TNF-expressing All cells caused fatal tumor growth only in 22% of the mice. 3 x 10(6) or 1.5 x 10(7). All cells produced progressive tumor growth and lethality in all male recipients. In sera of all the A11-cell-transplanted mice, biologically active TNF was detected shortly (4.5 h) after tumor inoculation (6 39 U/ml), decreasing to below detection level in the circulation by day 3. In recipients of 15 million A11 cells, circulating TNF reappeared and reached high levels (12-1000 U/ml) 3 to 7 weeks later, when the animals bore large tumors (14-23 mm). Generally, such mice became cachectic, severely anemic, hypothermic, and soon died. On account of calcium mobilization from bones, their serum Ca levels were high. Electron microscopy revealed severe liver damage, but there were no signs of chronic arthritis. These results suggest that ATS-treated mice xenotransplanted with TNF-gene-transfected A11 human tumor cells provide a new model for studying the pathophysiological and anti-tumor effects of TNF.

Phosphorylation of the 26 kDa TNF precursor in monocytic cells and in transfected HeLa cells.

Tumor necrosis factor (TNF) functions both as a soluble molecule and as a cell surface 26 kDa transmembrane protein, from which the soluble form is proteolytically derived. The 26 kDa TNF molecules isolated from 32P labeled HeLa cells that had been transfected with the cDNA of a partially cleavable TNF mutant were found labeled. Phosphorylated 26 kDa TNF molecules could also be isolated from human LPS stimulated monocytic Mono Mac 6. Phosphoaminoacid analysis revealed that the labeled phosphate is bound to serine residues. No label was found incorporated in soluble 17 kDa TNF, indicating that the phosphorylated residue(s) of membrane-associated TNF occur in the cytoplasmic portion of the molecule. Phosphorylation of the intracellular domain of the 26 kDa TNF molecules may play a role in the regulation of expression or proteolytic processing of TNF, modulate TNF bioactivity, or take part in intracellular signaling by cell-surface TNF.

Computed axial tomography scan demonstration of cerebral edema in eclampsia preceded by blindness.

The presence of cerebral edema in the occipital lobes are demonstrated by computed tomography (CT) in a patient presenting with eclampsia preceded by blindness at 36 weeks' gestation. After delivery by cesarean section, the patient's management included the use of mannitol and dexamethasone (Decadron) to reduce the cerebral edema. The patient's vision returned within 24 hours after delivery. A repeat CT scan confirmed the resolution of the cerebral edema.

CT of subacute sclerosing panencephalitis.

Subacute sclerosing panencephalitis is a progressive, frequently fatal slow virus infection of the brain attacking children and young adults and caused by measles virus. Computed tomography (CT) of the brain in 15 patients with this disease was normal in seven and abnormal in eight. CT demonstrated varying degrees of cortical atrophy associated with focal and multifocal low density lesions of the white matter. Two cases demonstrated low density lesions of the caudate nuclei. The CT lesions were seen in chronic cases and reflect the gliosis and atrophy that occur in this disease. Differential diagnoses include other viral infections and demyelinating and dysmyelinating diseases. Diagnostic laboratory evaluation includes serum and cerebrospinal fluid titers for measles antibodies, cerebrospinal fluid protein electrophoresis, electroencephalography, and cranial CT.

Clinical applications of combined cerebral angiograms and brain CT scans.

This paper illustrates the clinical value of a computer technique for superimposition of two different kinds of imaging procedures. The initial application was to overlay cerebral angiograms onto computed tomograms of the brain. Case material from three patients with intracerebral abnormalities is presented. The sum of information from the combined studies exceeds that from either study alone and has value for both diagnosis and treatment of head lesions.

Receptor-like properties of the 26 kDa transmembrane form of TNF.

Most members of the TNF family of proteins exist as transmembrane proteins with relatively long intracellular domains, and a number of them are involved in the ill-defined phenomenon of "reverse signaling". We have identified a putative nuclear localization signal in the cytoplasmic domain of TNF which proved to be functional in two assays. Western analysis identified an approximately 10 kDa peptide corresponding to the transmembrane and cytoplasmic domains of TNF after the proteolytic liberation of the 17 kDa, soluble form of TNF. This 10 kDa peptide was enriched in internal membranes and nuclear fractions of disrupted cells. Immune electron-microscopic studies proved its localization in transport vesicles and the nucleus. The nuclear transport of the intracellular segment of TNF resembles the signaling process through the Notch-type of receptors. Indeed, the presence of the 10 kDa peptide seems to influence the expression of another inflammatory cytokine, interleukin-1 beta. These findings suggest that the transmembrane form of TNF has receptor-like properties and its interaction with the receptors initiates a bidirectional signaling.

Delayed traumatic intracerebral hematomas. Report of three cases.

Three patients are reported who developed delayed intracerebral hematomas following closed head injuries. Two patients showed hematomas within 24 hours after a normal computerized tomogram. All three were treated surgically, and two had good results.

Some examples of balloon technology in neurosurgery.

Examples are presented of the use of a compression balloon to treat trigeminal neuralgia, of a dilating balloon to release a web obstruction of the internal carotid artery, of detachable balloons to seal carotid-cavernous and vertebral-venous fistulas, of a temporary occlusive balloon to aid in thrombogenic treatment of a giant aneurysm, and of a temporary occlusive balloon with double or triple-lumen capacity to assist in angiographic diagnosis and to provide reversible carotid occlusion.

beta-Endorphin-like immunoreactivity increases in human lumbar cerebrospinal fluid following routine metrizamide myelography.

Much interest has recently been focused on the possible role of the endogenous opiates in the perception of pain in humans. Several investigators have examined the levels of these substances in human cerebrospinal fluid (CSF) in attempts to identify the mechanisms by which electrical stimulation of the brain might induce analgesia. Most of these CSF samples were collected at the time of ventriculography or myelography. In the present study, the levels of beta-endorphin in the CSF of 22 patients undergoing myelography were examined before and after the injection of a contrast agent. beta-Endorphin increased an average of 356% (p less than 0.0005) 15 to 20 minutes following the injection of contrast material into the lumbar subarachnoid space. Thus, routine myelography may have a profound effect on the levels of beta-endorphin measured by radioimmunoassay in human CSF, and great care must be taken in interpreting the significance of changes seen in beta-endorphin levels in CSF collected from patients at the time of myelography or ventriculography. The effect of the injection of contrast material on beta-endorphin immunoreactivity must be distinguished from the postulated effects of any analgesia-inducing therapy.

A genetically modified recombinant tumor necrosis factor-alpha conjugated to the distal terminals of liposomal surface grafted polyethyleneglycol chains.

A genetically modified recombinant tumor necrosis factor (TNF)-alpha (rKRKTNF) was conjugated to the terminal carboxyl groups of liposome grafted polyethyleneglycol (PEG) chains. The long-circulating liposomes were composed of egg phosphatidylcholine, cholesterol (chol) and 7% carboxyl PEG-phosphatidylethanolamine. The conjugation efficiency of the genetically modified rKRKTNF under the conditions described in the text was approximately 55%. The biological activity of liposomal rKRKTNF, as tested with an in vitro cytotoxicity assay was reduced compared to the free, unconjugated rKRKTNF. In vivo biodistribution studies showed that conjugation of as little as 0. 13% of the grafted PEG chains resulted in a rapid elimination of the formulation from the blood stream. It is speculated that both non-selective conjugate chemistry and inherent recognition of the TNF by the components of the reticuloendothelial system (RES) are responsible for the short blood half life of the rKRKTNF-PEG-liposomes. The result suggest that conjugating a rapidly clearing recombinant cytokine to long-circulating liposomes provides little advantage in modifying the pharmacokinetic parameters of the cytokine.

Multiple meningiomas in the spinal canal.

Previous experimental and clinical observations suggest that radiation is a causative factor in the development of meningiomas. We report a rare case of three spinal meningiomas in a patient who had prior irradiation in the spinal region. The myelographic technique to evaluate multiple spinal canal tumors is outlined.