[Multisegmental spondylotic cervical myelopathy treated by longitudinal medial corporectomy without fusion]. / Mielopat a cervical espondil tica multisegmentaria tratada mediante corporectom a medial longitudinal sin fusión.

INTRODUCTION: The treatment of cervical spondylosis may be medical, surgical or both. Surgical treatment is indicated mainly in patients with untreatable pain, progressive neurological deficit and proven compression of the nerve roots and spinal cord. Corporectomy with a graft and fusion is one of the most widely used techniques carried out by neurosurgeons and orthopaedic surgeons using an anterior approach. PATIENTS AND METHODS: Between January 1993 and December 2000, six patients aged between 62 and 75 years, with a median age of 68.5 years, clinically and radiologically diagnosed as having multisegmental spondylotic cervical myelopathy, had surgical operations involving corporectomies without grafts. Nurick s scale was used to classify symptoms and radiological assessment was done before and after operation using plain Xrays, three dimensional computerized tomography and magnetic resonance. In one case one corporectomy was done, in two cases two and in three cases three. RESULTS: The patients follow up varied from 3 months to seven years. In four patients symptoms improved, two became stable and in no case was there any radiological sign of vertebral instability. As far as we know after reviewing the literature, this is the first time that this technique has been published. CONCLUSIONS: Corporectomy without grafting is a new, simple, effective technique for the treatment of multisegmental spondylotic cervical myelopathy.

Mielopatía cervical espondilótica multisegmentaria tratada mediante corporectomía medial longitudinal sin fusión / Multisegmental spondylotic cervical myelopathy treated by longitudinal medial corporectomy without fusion

Introducción. El tratamiento de la espondilosis cervical puede ser médico, quirúrgico, o ambos. El tratamiento quirúrgico está indicado, fundamentalmente, para aquellos pacientes con dolor intratable, déficit neurológico progresivo, y compresión documentada de las raíces y del cordón medular. La corporectomía con injerto y fusión constituye una de las técnicas quirúrgicas más ampliamente realizadas por vía anterior por neurocirujanos y ortopedas. Pacientes y métodos. Entre enero de 1993 y diciembre del 2000, seis pacientes, en edades comprendidas entre los 62 y 75 años (media, 68,5 años), diagnosticados clínica y radiológicamente de una mielopatía cervical espondilótica multisegmentaria, fueron intervenidos quirúrgicamente mediante sendas corporectomías sin injerto. La escala de Nurick se utilizó para categorizar los síntomas, y la valoración radiológica se realizó antes y después de la cirugía, mediante rayos x simple, tomografía computadorizada tridimensional y resonancia magnética. Una corporectomía se practicó en un caso, dos corporectomías en dos casos y una triple corporectomía se realizó en tres pacientes. Resultados. El seguimiento de los pacientes osciló entre tres meses y siete años; cuatro pacientes mejoraron de sus síntomas, dos se estabilizaron y en ninguno de los casos se detectaron signos radiológicos de inestabilidad vertebral. Para nuestro conocimiento, y después de haber revisado la literatura, ésta es la primera publicación que habla de esta técnica. Conclusión. La corporectomía sin injerto es una técnica nueva, sencilla y eficaz para el tratamiento de la mielopatía cervical espondilótica multisegmentaria (AU)

HV(MNE), a novel lymphocryptovirus related to Epstein-Barr virus, induces lymphoma in New Zealand White rabbits.

HV(MNE) is a novel Epstein-Barr (EBV)-like virus isolated from a Macaca nemestrina with CD8(+) T-cell mycosis fungoides-cutaneous T-cell lymphoma. Here it is demonstrated that intravenous inoculation of irradiated HV(MNE)-infected T cells or cell-free virus from the J94356(PBMC) cell line in New Zealand White rabbits results in seroconversion to the viral capsid antigen (VCA) of EBV; all animals that seroconverted to VCA developed malignant lymphoma within months of inoculation. In contrast, control rabbits, inoculated with heat-inactivated culture supernatants from the same cell line, failed to seroconvert to VCA and did not develop disease. Disseminated lymphoma cells of mixed origin were detected in most vital organs, including the spleen, liver, lungs, kidneys, and heart of the affected rabbits. Neoplastic infiltrates were also observed in lymph nodes, thymus, skin, and subcutaneous tissues. HV(MNE) DNA and EBV-like RNA expression was demonstrated in the lymphomatous organs and in 2 transformed T-cell lines, one established from the lymph node and the other from the blood of the 2 lymphomatous animals. Analysis of one of these T-cell lines demonstrated the persistence of HV(MNE) DNA, expression of an LMP1-like protein, and acquisition of interleukin-2 independence, and constitutive activation of the Jak/STAT pathway. Thus, HV(MNE) in rabbits provides a valuable animal model for human T-cell lymphoma whereby genetic determinants for T-cell transformation by this EBV-like animal virus can be studied.

The HTLV-I orfI protein is recognized by serum antibodies from naturally infected humans and experimentally infected rabbits.

The mechanism of T-cell transformation by human T-cell lymphotropic virus type I (HTLV-I), though not completely understood, appears to involve the interactions of several viral and cellular proteins. One of these viral proteins, p12(I), encoded by HTLV-I orfI, is a weak oncogene that binds the 16-kDa subunit of the vacuolar ATPase and interacts with the immature beta and gamma(c) chains of the IL-2 receptor. We have expressed the singly spliced orfI cDNA in the baculovirus system and used the recombinant protein as a tool to assess the presence of antibodies in naturally or experimentally infected hosts. In addition, rabbit antisera were raised against various p12(I) synthetic peptides and used to identify three antigenic regions within p12(I), one between the two putative transmembrane regions of p12(I) and two at the carboxy-terminus of the protein. More importantly, sera from a naturally infected human (1 of 32) and experimentally infected rabbits (9 of 20) recognized the rp12(I), demonstrating orfI expression and immunogenicity in vivo. Taken together these data provide the first evidence of orfI expression during HTLV-I infections.

A novel Epstein-Barr virus-like virus, HV(MNE), in a Macaca nemestrina with mycosis fungoides.

Epstein-Barr virus (EBV) infection of humans has been associated with the development of lymphoid malignancies mainly of B-cell lineage, although occasionally T-cell lymphomas have been reported. We describe here the characterization of a novel EBV-like virus (HV(MNE)) isolated from a simian T-cell lymphotropic virus type I/II (STLV-I/II) seronegative pigtailed macaque (Macaca nemestrina) with a cutaneous T-cell lymphoma. Immunohistochemistry studies on the skin lesions demonstrated that the infiltrating cells were of the CD3(+)/CD8(+) phenotype. Two primary transformed CD8(+) T-cell lines were obtained from cultures of peripheral blood mononuclear cells (PBMC) and skin, and, with time, both cell lines became interleukin-2-independent and acquired the constitutive activation of STAT proteins. Polymerase chain reaction analysis of the DNA from the cell lines and tissues from the lymphomatous animal demonstrated the presence of a 536-bp DNA fragment that was 90% identical to EBV polymerase gene sequences, whereas the same DNA was consistently negative for STLV-I/II sequences. Electron microscopy performed on both cell lines, after sodium butyrate treatment, showed the presence of a herpes-like virus that was designated HV(MNE) according to the existing nomenclature. In situ hybridization studies using EBV Epstein-Barr viral-encoded RNA probes showed viral RNA expression in both CD8(+) T-cell lines as well as in the infiltrating CD8(+) T cells of skin-tissue biopsies. Phylogenetic analysis of a 465-bp fragment from the polymerase gene of HV(MNE) placed this virus within the Lymphocryptovirus genus and demonstrated that HV(MNE) is a distinct virus, clearly related to human EBV and other EBV-like herpesviruses found in nonhuman primates.

Limiting amounts of p27Kip1 correlates with constitutive activation of cyclin E-CDK2 complex in HTLV-I-transformed T-cells.

Human T-cells immortalized (interleukin-2 [IL-2] dependent) by the human T-cell lymphotropic/leukemia virus type I (HTLV-I), in time, become transformed (IL-2 independent). To understand the biochemical basis of this transition, we have used the sibling HTLV-I-infected T-cell lines, N1186 (IL-2 dependent) and N1186-94 (IL-2 independent), as models to assess the responses to antiproliferative signals. In N1186 cells arrested in G1 after serum/interleukin-2 (IL-2) deprivation, downregulation of the cyclin E-CDK2 kinase activity correlated with decreased phosphorylation of CDK2 and accumulation of p27Kip1 bound to the cyclin E-CDK2 complex, as seen in normal activated PBMCs (peripheral blood mononuclear cells). In contrast, N1186-94 cells failed to arrest in G1 upon serum starvation, displayed constitutive cyclin E-associated kinase activity, and, although CDK2 was partially dephosphorylated, the amount of p27Kip1 bound to the complex did not increase. This observation, extended to two other IL-2-dependent as well as to three IL-2-independent HTLV-I-infected T-cell lines, suggests that the lack of cyclin E-CDK2 kinase downregulation found in the late phase of HTLV-I transformation may correlate with insufficient amounts of p27Kip1 associated with the cyclin E-CDK2 complex. Reconstitution experiments demonstrated that the addition of p27Kip1 to lysates from N1186-94 starved cells resulted in the downregulation of cyclin E-associated kinase activity supporting the notion that the unresponsiveness of the cyclin E-CDK2 complex to growth inhibitory signals may be due to inadequate amounts of p27Kip1 assembled with the complex in HTLV-I-transformed T-cells. In fact, the amount of p27Kip1 protein was lower in most HTLV-I-transformed (IL-2-independent) than in the immortalized (IL-2-dependent) HTLV-I-infected T-cells. Furthermore, specific inhibitors of the phosphatidylinositol 3-kinase (P13K) induced an increase of p27Kip1 protein levels, which correlated with G1 arrest, in both IL-2-dependent and IL-2-independent HTLV-I-infected T-cells. Altogether, these results suggest that maintaining a low level of expression of p27Kip1 is a key event in HTLV-I transformation.

Sites of recombinant adeno-associated virus integration.

Adeno-associated virus (AAV), a defective parvovirus, is considered a promising vector for the delivery of therapeutic genes to cells. Both wild-type and recombinant AAV display a wide tropism and integrate into the host genome, in the absence of helper virus, establishing a latent infection. A unique characteristic of wild-type AAV and a potential advantage for use as a delivery system for gene therapy is the site-specific integration of wild-type virus within a small region of chromosome 19, 19q13.3-qter (AAVS1), in up to 85% of cell lines infected with the virus. Although recombinant AAVs, containing only the inverted terminal repeats of wild-type virus, can integrate efficiently into the host genome, specificity for the AAVS1 site appears to be lost. To address this question, the integration characteristics of two recombinant AAVs lacking the rep and cap genes in HeLa cells were examined. Analysis of Southern blots indicated that none of twenty-six cell clones generated after infection with either one of the recombinant AAVs demonstrated integration within the AAVS1 locus on chromo-some 19. Analysis of five of the cell lines by fluorescent chromosome in situ hybridization confirmed the loss of chromosome 19 specificity. Each integration site mapped near a known fragile site and/or location of a proto-oncogene or growth regulatory gene. Retention of site-specific integration of wild-type AAV will require the inclusion of additional AAV-specific sequences within the recombinant vectors.

The tax gene sequences form two divergent monophyletic lineages corresponding to types I and II of simian and human T-cell leukemia/lymphotropic viruses.

Evolutionary associations of human and simian T-cell leukemia/lymphotropic viruses I and II (HTLV-I/II and STLV-I/II) are inferred from phylogenetic analysis of tax gene sequences. Samples studied consisted of a geographically diverse assemblage of viral strains obtained from 10 human subjects and 20 individuals representing 12 species of nonhuman primates. Sequence analyses identified distinct substitutions, which distinguished between viral types I and II, irrespective of host species. Phylogenetic reconstruction of nucleotide sequences strongly supported two major evolutionary groups corresponding to viral types I and II. With the type I lineage, clusters were composed of strains from multiple host species. A genetically diverse, monophyletic lineage consisting of eight new viral strains from several species of Asian macaques was identified. The second lineage consisted of a monophyletic assemblage of HTLV-II/STLV-II strains from Africa and the New World, including an isolate from a pygmy chimp (Pan paniscus) as an early divergence within the lineage. High levels of genetic variation among strains from Asian STLV-I macaque suggest the virus arose in Asia. Evidence of the origin of the type II virus is less clear, but diversity among HTLV-II variants from a single isolated population of Mbati villagers is suggestive but not proof of an African origin.

Cellular CD44S as a determinant of human immunodeficiency virus type 1 infection and cellular tropism.

CD4 is the predominant cell membrane protein that binds human immunodeficiency virus type 1 (HIV-1) gp120 and facilitates HIV-1 infection, but other membrane-associated molecules may be involved in determining HIV-1 cellular infection. Our prior work had suggested that CD44, the transmembrane receptor for hyaluronan, might play a role in the infection of mononuclear phagocytes with HIV-1. In the present work, we have used cells of the CD4-positive, CD44-negative human T-lymphoblast cell line Jurkat to study the role of CD44 in HIV-1 infection and tropism. Cells were transfected with cDNA for the standard (S, or hematopoietic) CD44 isoform CD44S or the epithelial isoform CD44E. The resultant lines expressed appropriate CD44S or CD44E mRNA and protein. While the parent Jurkat cells, those transfected with vector alone, and those transfected with CD44E could be productively infected with only the lymphocytotropic strain HIV-1-LAI, cells transfected with CD44S were rendered susceptible to productive infection with the monocytotropic strains HIV-1-BaL and HIV-1-ADA. Also, CD44S-transfected cells displayed higher levels of infection with HIV-1-LAI than did the other transfected Jurkat cells. The transfected cell line cells all had comparable growth rates and expressed similar levels of the membrane antigens CD4, CD7, major histocompatibility complex (MHC) class I, MHC class II, and CD11a, while levels of CD3 were slightly higher in cells transfected with vector alone and in one of the clones transfected with CD44S. Hyaluronan binding was increased in cells transfected with either CD44S or CD44E. Mouse NIH 3T3 fibroblasts transfected with human CD4, human CD44S, or both human CD4 and CD44S displayed the appropriate antigens, but they could not be productively infected with lymphocytotropic or monocytotropic strains of HIV-1. The results indicate that in human leukocytes, CD44S is an important determinant of HIV-1 productive infection and may be involved in viral cellular tropism.

Inhibition of HIV type 1 infection of mononuclear phagocytes by anti-CD44 antibodies.

Cellular CD4 is the primary membrane molecule that binds HIV-1 through interaction with viral gp120. Membrane glycolipids and cell adhesion molecules have also been noted to be involved in the interaction of HIV-1 with cells and in syncytium formation in infected cells. The purpose of this study was to determine the role of the cell adhesion molecule CD44 in HIV-1 infection of cells. Both normal blood monocytes and lymphocytes expressed CD44 as determined by flow cytometry using the anti-CD44 antibody A3D8. Anti-CD44 monoclonal antibodies A3D8, A1G3, and 5F12 [ascites, purified IgG, and F(ab')2] inhibited infection of monocytes and peritoneal macrophages with HIV-1-BaL and HIV-1-ADA, but had no effect on HIV-1-IIIB infection of mitogen-stimulated lymphocytes, or cells of a T lymphocyte line. CD44 monoclonal antibodies were not toxic for monocytes, and the observed inhibitory effect of CD44 monoclonal antibodies was not dependent on complement. These results suggest that CD44 may be a determinant of HIV-1 infection of mononuclear phagocytes in vitro.

Dosing considerations for oral acyclovir following neonatal herpes disease.

Herpes simplex virus lesions recur in 8-30% of infants who receive a course of parenteral antiviral therapy for an initial infection. Long-term acyclovir is used by some clinicians to prevent recurrent Herpes simplex disease. We describe nine infants who were treated with doses of oral acyclovir which were chosen to achieve 2-h post-plasma concentrations of > or = 2 micrograms/ml. Eight infants had Herpes simplex encephalitis and one had multiple recurrences of dermal and ocular disease. The target plasma concentration was chosen in order to attain acyclovir cerebrospinal fluid distribution (< or = 50% plasma) for an estimated ID30 of Herpes simplex II strains of 0.1-0.5 microgram/ml. One of nine patients failed to achieve the target plasma acyclovir concentration. One of nine patients developed symptomatic recurrence of the central nervous system disease and none of the remaining eight patients experienced recognized dermal or neurologic recurrence of Herpes simplex disease. Renal and neurologic status were routinely monitored and no signs of acyclovir toxicity were observed. Plasma concentration of acyclovir > or = 2 micrograms/ml may be achieved with average oral doses of 1340 mg/m2/dose (1000-1740 mg/m2/dose) given at 12-h intervals.

Neopterin production by HIV-1-infected mononuclear phagocytes.

Neopterin is a pteridine produced by human mononuclear phagocytes, usually in response to interferon-gamma (IFN-gamma) stimulation. Increasing serum levels of neopterin correlate with clinical progression to AIDS in HIV-infected people, but the factors that contribute to these elevated levels are not established. We performed in vitro experiments to investigate the possibility that HIV-1 infection of mononuclear phagocytes directly induces enhanced neopterin production. We found that HIV-1-infected monocytes and peritoneal macrophages produced neopterin in quantities similar to amounts produced by uninfected cells. The HIV-infected cells responded to stimulation with IFN-gamma as well as uninfected cells, with a 6- to 12-fold increase in neopterin production. We conclude that elevated serum levels of neopterin in HIV-infected individuals are not caused by HIV-1 infection of mononuclear phagocytes but may be a result of the normal response to mononuclear phagocytes to increased levels of IFN-gamma.

Separation and concentration of schizonts of Plasmodium falciparum by Percoll gradients.

A technique for the separation of schizonts of Plasmodium falciparum is described. The different stages of the asexual cell cycle of the parasite were positioned according to their density in a continuous gradient of Percoll. Young trophozoites coincided with erythrocytes in a broad band corresponding to densities from 1.075 to 1.100 g/ml, whereas schizonts were concentrated at a density approximating 1.062 g/ml. The viability of the parasites was unimpaired by this procedure. Young trophozoites and schizonts continued their normal life cycle when cultured after the separation procedure. The percentage of recovery was high, reaching 80% of the initial quantity. Possible applications of the technique are discussed.