Peripheral neuropathy associated with anti-myelin basic protein antibodies in a woman vaccinated with rubella virus vaccine.

Active immunisation with rubella vaccine has not been commonly associated with neurological complications. We report the case of a 23-year-old woman who developed a mild, distal demyelinating neuropathy after immunisation with the live attenuated RA 27/3 rubella strain. Post-immunisation immunologic studies carried over 24 months showed the presence of antibodies to the RV proteins, particularly to the capsid antigen, and to the myelin basic protein (MBP). A similarity between a C antigen motif and a sequence of the MBP was found by computer analysis. The cross-reactivity was confirmed by immunising mice with a synthetic peptide derived from the MBP, which developed a strong humoral response to RV and MBP. This finding raises the possibility that a virus-induced immune response could lead to an autoaggressive reaction responsible for demyelination.

Long-lasting protection by live attenuated simian immunodeficiency virus in cynomolgus monkeys: no detection of reactivation after stimulation with a recall antigen.

The infection of cynomolgus monkeys with an attenuated simian immunodeficiency virus (SIV) (C8) carrying a deletion in the nef gene results in a persistent infection associated with an extremely low viral burden in peripheral blood mononuclear cells. The aim of this study was to determine (1) the breadth of the protection after repeated challenges of monkeys with SIV homologous strains of different pathogenicity, (2) the genotypic stability of the live virus vaccine, (3) whether the protection might depend on cellular resistance to superinfection, and (4) whether immunogenic stimuli such as recall antigens could reactivate the replication of the C8 virus. To address these goals, the monkeys were challenged at 40 weeks after C8 infection with 50 MID50 of cloned SIVmac251, BK28 grown on macaque cells. They were protected as indicated by several criteria, including virus isolation, anamnestic serological responses, and viral diagnostic PCR. At 92 weeks after the first challenge, unfractionated peripheral blood mononuclear cells from protected monkeys were susceptible to the in vitro infection with SIVmac32H, spl. At 143 weeks after C8 infection, the four protected monkeys were rechallenged with 50 MID50 of the pathogenic SIVmac32H, spl grown on macaque cells. Once again, they were protected. The C8 virus remained genotypically stable, and depletion of CD4(+) cells was not observed during approximately 3 years of follow-up. In contrast, it was found that the infection with SIVmac32H, spl induced CD4(+) cell depletion in three of three control monkeys. Of importance, stimulation with tetanus toxoid, although capable of inducing specific humoral and T cell proliferative responses, failed to induce a detectable reactivation of C8 virus.

Detection of infectious simian immunodeficiency virus in B- and T-cell lymphomas of experimentally infected macaques.

An increasing frequency of malignant lymphomas occurs among patients infected by human immunodeficiency virus. Because of the close similarities to human malignancies, we used a nonhuman primate model to study the pathogenesis of simian immunodeficiency virus (SIV)-associated malignancies. Specifically, we investigated (1) the presence of the SIV genome in tumor cells, (2) the presence of coinfecting viruses, and (3) the presence of a rearrangement of the immunoglobulin and c-myc genes. We observed 5 cases of non-Hodgkin's lymphomas (4 of B- and 1 of T-cell origin) among 14 SIV-infected cynomolgus monkeys. No c-myc translocation was observed in the tumors, whereas B-cell lymphomas were characterized either by a monoclonal (in 2 of 4) or by an oligoclonal (in 2 of 4) VDJ rearrangements of the immunoglobulin heavy chain gene. Molecular, biological, and immunological analyses did show the presence of infectious SIV in the tumor cells of 1 T-cell and 2 oligoclonal B-cell lymphomas. Neither Simian T-lymphotropic nor Epstein-Barr viruses were detectable, whereas Simian herpes virus Macaca fascicularis-1 was detectable at a very low copy number in 3 of 4 B-cell lymphomas; however, only 1 of these also harbored the SIV genome. These results support the possibility that SIV may be directly involved in the process of B or T lymphomagenesis occurring in simian acquired immunodeficiency syndrome.

Effects of lethal irradiation and cyclosporin A treatment on the growth and tumoricidal activity of a T cell clone potentially useful in cancer therapy.

The TALL-104 cell line, originally derived from a patient with T cell leukemia, can be maintained indefinitely in culture in the presence of interleukin-2 (IL-2) and is endowed with a highly potent major-histocompatibility-complex (MHC)-non-restricted tumoricidal activity both in vitro and in animal models. The present study analyzes in detail the short- and long-term effects of irradiation and cyclosporin A (CsA) treatment on the growth and tumoricidal function of this T cell clone as compared to polyclonal lymphokine-activated killer (LAK) cell preparations from healthy donors. DNA and RNA syntheses by both TALL-104 and LAK cells were irreversibly arrested a few hours after irradiation with 40 Gy. However, 4-h 51Cr-release assays, performed on different days (day 1 to day 7) after irradiation, showed that the cytotoxic efficiency of TALL-104 cells against hematopoietic and solid tumor targets was only modestly reduced, whereas that of LAK cells was severely inhibited. Moreover, the cytotoxic responses to recombinant human IL-2 and IL-12, measured 18 h after irradiation and cytokine addition, were normal in the case of TALL-104 cells but were abolished in the case of LAK cells. Co-culture of IL-2- or IL-12-preactivated TALL-104 cells with a tumor target for 5 days in the absence of cytokines resulted in a lower efficiency of lysis, as compared to the non-irradiated effectors, especially if the initial stimulus was IL-12. These findings suggest the requirement of multiple cytokine stimulation for optimal expression of tumoricidal activity by lethally irradiated TALL-104 cells. CsA, while abrogating TALL-104 cell proliferation at the low dose of 0.5 microgram/ml, inhibited their cytotoxic function marginally only at high doses (100 micrograms/ml). By contrast, CsA reduced dose-dependently the cytotoxicity of LAK cells starting at very low doses (0.5 microgram/ml). CsA did not impair the ability of TALL-104 and LAK cells to produce interferon (IFN) gamma, tumor necrosis factor (TNF) alpha, and granulocyte/macrophage-colony-stimulatory factor (GM-CSF) in response to IL-2, IL-12, or tumor targets. Irradiation reduced drastically IFN gamma production by LAK, but not TALL-104 cells; release of TNF alpha and GM-CSF by either type of effector was inhibited by 10%-50%, depending on the stimulus. The high resistance and immunosuppressive drugs renders tis immortal T cell clone a potentially safe and effective reagent for new adoptive-transfer approaches to cancer in MHC-incompatible recipients.

Reversal of acute myelogenous leukemia in humanized SCID mice using a novel adoptive transfer approach.

Advanced human malignancies cannot be permanently cured by adoptive transfer of autologous lymphokine-activated killer (LAK) cells. Moreover, administration of high doses of IL-2 to maintain LAK activity in vivo is associated with severe toxicity. In this study, we tested the anti-tumor efficacy of a uniquely potent MHC non-restricted human killer T cell clone (TALL-104) in humanized severe combined immunodeficient (SCID) mice bearing acute myelogenous leukemia (AML). We show that, in appropriate experimental conditions, TALL-104 cells could effectively reverse the aggressive growth of the myelomonocytic leukemia cell line U937 in SCID mouse tissues, leading to complete abrogation of AML. A single transfer of TALL-104 cells at the time of tumor challenge in combination with recombinant human (rh) IL-12 (1 microgram/d) prolonged significantly the life of the mice. However, eradication of leukemia, as monitored both clinically and by PCR, was achieved by repeated injection of the effectors at close intervals. Complete cure was obtained also upon transfer of lethally irradiated (non-proliferating) TALL-104 cells together with low doses of rh IL-2 (100 U/d). Most notably, of the mice that received multiple transfers of TALL-104 cells without cytokines in an advanced disease stage, 50% were clinically cured, and 50% survived significantly longer. The potential of TALL-104 cells as an effective and safe leukemia purging agent is discussed.

Expression of recombinant E2 and C proteins of rubella virus in insect cells.

We have constructed a recombinant baculovirus expressing the rubella virus E2 (42-45 KDa) and C (34 KDa) proteins. Sf9 cells infected with recombinant virus were able to synthesize and process the two proteins coded by a unique precursor gene. By immunoblot and immunoprecipitation analysis with polyclonal and monoclonal antibodies, a precursor polyprotein (66 KDa) and two other proteins migrating with an apparent molecular weight of 42 KDa and 36 KDa were recognized as E2 glycoprotein and C protein, respectively. The recombinant E2 protein appeared to be glycosylated since it was susceptible to tunicamycin. The results indicate that the RV polyprotein coding for E2 and C is expressed and proteolytically cleaved in insect cells. This baculovirus expression system provides a useful alternative approach for the production of rubella virus antigens and should allow the purification of large quantities of the RV proteins for further biochemical and immunological studies.

Selective inhibition of proliferation in v-abl- and bcr-abl-transformed cells by a nucleoside analog.

The nucleoside analog acyclovir (9-[2-hydroxy-ethoxy)methyl]guanine or acycloguanosine; ACV) inhibited the in vitro transformation of NIH 3T3 cells by Abelson murine leukemia virus and the proliferation of abl- and bcr-abl-transformed hemopoietic murine cell lines. This effect is selective since ACV at the same concentration had no effect on the src and Ha-ras transformation of NIH 3T3 cells or on the proliferation of hemopoietic cells transformed by those oncogenes. The inhibitory effect on proliferation of abl-transformed cells correlated with the extent of ACV triphosphate formation and incorporation into cellular DNA that was greater than that in normal or other oncogene-transformed cells. The increased ACV triphosphate formation might be due to a higher level of 5'-nucleotidase, the enzyme responsible for trace levels of ACV phosphorylation in uninfected cells.

Role of membrane phospholipids and glycolipids in the Vero cell surface receptor for rubella virus.

Membrane receptors for rubella virus (RV) in Vero cells were studied by means of two different approaches: (i) by enzyme treatment of the whole cell membrane and (ii) by testing the ability of isolated plasma membrane molecules to compete with cells for virus binding. The replication of RV was studied with both indirect immunofluorescence assay and molecular hybridization techniques. Phospholipases A2 and C digestion of cells greatly reduced the infectivity by the virus, pointing towards the involvement of lipid structures as receptor sites for RV. Furthermore, susceptibility of Vero cells to virus infection was also reduced after beta-N-acetyl-D-glucosaminidase, alpha-glucosidase and beta-galactosidase treatment, suggesting that carbohydrate residues may participate in a complex cellular receptor structure for RV. When the major membrane lipids were examined separately for their ability to inhibit viral infectivity, several phospholipids (phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylcholine, sphingomyelin) and glycolipids (gangliosides, lactosylceramide, cerebroside sulphate) showed a strong neutralizing activity, confirming the role of membrane lipid moiety in the cell surface receptor for RV.

Binding sites for rubella virus on erythrocyte membrane.

To identify molecule(s) with the properties of rubella virus (RV) receptor, goose erythrocyte membranes were isolated and tested for their ability to complete with whole cells for viral binding and fusion. Solubilized membranes showed a dose-dependent inhibiting activity on either rubella virus attachment or its fusion with erythrocytes at acidic pH. The inhibitory activity was enhanced by trypsin and neuraminidase, and inactivated by phospholipase A2 digestion, pointing towards the involvement of lipid structures as receptor sites for RV. After isolation of the different membrane components, only the lipid moiety, specifically phospholipids and glycolipids, was found to inhibit viral biological activities. When the major membrane lipids were examined separately, phosphatidylserine and cerebroside sulfate showed a strong inhibiting activity on viral hemagglutination and subsequent hemolysis. The capacity of several pure phospholipids (phosphatidylinositol, phosphatidylcholine and sphingomyelin) to inhibit the hemolysis but not the binding of the virus to the erythrocytes indicated that different membrane lipid components are involved in the attachment and the fusion step. Enzymatic and chemical modifications of whole erythrocytes confirmed the role of membrane lipid molecules in the cell surface receptor for RV.

Cloning and nucleotide sequence of a mouse erythrocyte beta-spectrin cDNA.

A rabbit monospecific antibody for mouse beta-spectrin was used to screen a mouse anemic spleen cDNA expression library. A mouse beta-spectrin cDNA clone was isolated and identified by its ability to make mouse beta-spectrin-like antigens in Escherichia coli. This clone was used to probe total RNA from various mouse tissues. Anemic spleen RNA showed two strongly hybridizing RNA species of approximately 6 and 8 kb. Two very faintly hybridizing bands of about 6 kb and 10 kb could also be seen in total mouse brain RNA. All of these bands could be detected after hybridization under both stringent and nonstringent conditions. This suggests that erythroid beta-spectrin may also be expressed in the brain. No bands could be detected in kidney, liver, or spleen RNA. Southern blot analysis of mouse genomic DNA showed a single hybridizing band after digestion with several restriction endonucleases even under nonstringent conditions. Nucleotide sequencing of the cDNA insert revealed almost complete identity between the N-terminus of the deduced amino acid sequence of the cDNA clone and the C-terminal 15 amino acids of a peptide derived from the beta-8 repeat unit of human erythrocyte beta-spectrin. The deduced amino acid sequence contained most of the conserved amino acids characteristic of the 106 amino acid repeat unit first found in human alpha-spectrin and thus provides the first evidence for a complete 106 amino acid repeat unit structure in beta-spectrin.

Assignment of mouse beta-spectrin gene to chromosome 12.

The structural gene for the beta-subunit of the mouse erythrocyte spectrin, hereinafter designated as Sp-b, was assigned to the mouse chromosome 12. This assignment was made by Southern analysis of genomic DNA from mouse X Chinese hamster hybrid cells using cloned mouse erythrocyte beta-spectrin cDNA as a probe. In the PstI-digested genomic hamster cell DNA a single band of 2.0 kb was detected, whereas PstI-digested mouse DNA gave a band of 4.2 kb, when probed with the mouse erythroid beta-spectrin cDNA clone. This allowed us to analyze a panel of mouse X Chinese hamster somatic cell hybrids to map this gene to chromosome 12. Interestingly, this assignment is different from that observed for the alpha-subunit of spectrin, which has been mapped to chromosome 1 in mouse. These results serve as a basis for further genetic characterization of the mouse hemolytic anemias.

Cloning of rabies virus matrix protein mRNA and determination of its amino acid sequence.

A cDNA clone of mRNA for rabies virus matrix (M) protein has been identified. The clone hybridizes to an mRNA species from rabies virus-infected cells, whose size correlates to the size of the M protein in rabies virions, and selects an mRNA that translates into a polypeptide corresponding in size to M protein. The nucleotide sequence of the cloned cDNA was determined and from this a complete amino acid sequence for M protein was deduced. The deduced sequence of 202 amino acids bears no detectable sequence homology with vesicular stomatitis virus M protein although these proteins may share functional homology.

Cloning and structural characterization of a human non-erythroid band 3-like protein.

Polypeptides which are immunologically related to the erythrocyte anion transport protein have been identified in a variety of non-erythroid cells. We describe two cDNA clones encoding a human non-erythroid band 3 protein (HKB3) and the mouse erythrocyte band 3 (MEB3) and show that these proteins are structurally similar. Comparison of the predicted amino acid sequences from HKB3 and MEB3 reveals a high degree of sequence homology (71%) and conservation of the overall topography of the transmembrane domain. Similar levels of homology are also observed in comparisons with published amino acid sequence from the human erythrocyte band 3. In addition, specific residues which have been demonstrated to be involved in erythroid anion transport are conserved in HKB3, suggesting that this non-erythroid band 3 protein functions in this respect. Although protein sequence homology within the cytoplasmic domain is considerably lower (35%), three specific regions in HKB3 are conserved, one of which may represent an ankyrin binding site. Northern blot analysis reveals transcripts that cross-hybridize with the HKB3 cDNA in a variety of non-erythroid cell lines but not in cells of erythroid lineage.

Detection and characterization of a mouse alpha-spectrin cDNA clone by its expression in Escherichia coli.

A cloned segment of mouse alpha-spectrin mRNA has been identified by immunological techniques. Double-stranded cDNA derived from spleens of anemic mice was introduced into a bacterial expression vector, pUC, and transformed Escherichia coli colonies were screened by using an antiserum to erythrocyte membrane ghost proteins. Of 17 positive colonies, 2 bound antibody to mouse spectrin, and these 2 colonies contained 750-base-pair inserts that cross-hybridized. Transfer of the 750-base-pair insert to an expression vector containing the PL promoter of phage lambda produced larger amounts of peptides that were bound by antibody to mouse spectrin. The spectrin-like peptides made in E. coli elicited antibody that reacted only with the alpha-spectrin subunit of erythrocyte membranes. This clone will be useful for the study of the structure and expression of the spectrin gene, particularly in understanding the role of spectrin in human inherited hemolytic anemias.

Sequence comparison of human and murine erythrocyte alpha-spectrin cDNA.

The results of hybridization analyses using cDNA probes for mouse and human alpha-spectrin mRNA indicate that a single gene encodes the alpha-subunit of erythrocyte spectrin. Sequencing of the cDNA clones showed that they code for 370 amino acids (aa) covering three repeat domains close to the C terminus of alpha-spectrin. The cloned cDNAs will now permit the isolation of the alpha-spectrin gene and should lead to the characterization of the genetic aspects in human hereditary anemias in which alpha-spectrin has been characterized as the site of the molecular defect.

Defective interferon-gamma production in ataxia-telangiectasia.

The in vitro production of interferon-alpha and -gamma (IFN) by peripheral blood mononuclear cells from four patients with ataxia-telangiectasia was compared to that of healthy controls. Normal values of IFN-alpha were obtained in all cases. However, patients with ataxia-telangiectasia showed a great reduction or absence of IFN-gamma production after induction with either staphylococcal enterotoxin B or galactose oxidase. This defect was accompanied by the absence of secretion of another lymphokine, namely, interleukin 2 (IL-2), in one case. Lymphoproliferative response to phytohemagglutinin (PHA) was severely depressed in all patients. Near normal values of T lymphocytes were found, but the ratio of OKT4+/OKT8+ subsets was reduced in most patients, due to a decrease of OKT4+ lymphocytes. Deficiency of IFN-gamma may contribute to the abnormalities of immune functions and immunoregulation observed in ataxia-telangiectasia, and it may represent an additional cause of the high incidence of viral infections and neoplasia in this disease.

Modulation of hemoglobin synthesis in K-562(S) cells treated with interferons.

Treatment of K-562(S) cells with human interferons (HulFN) alpha, beta, or gamma results in a modulation of hemoglobin (Hb) production. When K-562(S) cells, "induced" with butyric acid or hemin, are given low dosages of all three types of IFN, the percent benzidine-positive cells doubles. Treatment with low doses of IFN causes the acceleration and increased production of those Hb types (as shown by Cellogel analysis) that are already synthesized either in untreated or in "induced" cultures. These results were confirmed by using pure HulFN-beta, and were abrogated, for HulFN-alpha, in presence of its specific antiserum. In contrast, cultures of K-562 cells treated with either inducer and given more than 10(4) IU/ml of alpha- or beta-IFN show a dose-dependent decrease of hemoglobinization in the absence of cell death. The inhibitory effect was reversible upon removal of IFN and culture reseeding in IFN- and inducer-free medium. The significance of both sets of data is strengthened by the pronounced heterogeneity of K-562S cells with respect to their sensitivity to IFN treatment as evaluated by the establishment of the antiviral state. Apparently, one cell out of two is sensitive to IFN, which suggests that the magnitude of IFN effects described here may be larger than it appears to be from the data taken at face value.