GvL effects in T-prolymphocytic leukemia: evidence from MRD kinetics and TCR repertoire analyses.

Allogeneic stem cell transplantation (alloSCT) is used for treating patients with T-prolymphocytic leukemia (T-PLL). However, direct evidence of GvL activity in T-PLL is lacking. We correlated minimal residual disease (MRD) kinetics with immune interventions and T-cell receptor (TCR) repertoire diversity alterations in patients after alloSCT for T-PLL. Longitudinal quantitative MRD monitoring was performed by clone-specific real-time PCR of TCR rearrangements (n=7), and TCR repertoire diversity assessment by next-generation sequencing (NGS; n=3) Although post-transplant immunomodulation (immunosuppression tapering or donor lymphocyte infusions) resulted in significant reduction (>1 log) of MRD levels in 7 of 10 occasions, durable MRD clearance was observed in only two patients. In all three patients analyzed by TCR-NGS, MRD responses were reproducibly associated with a shift from a clonal, T-PLL-driven profile to a polyclonal signature. Novel clonotypes that could explain a clonal GvL effect did not emerge. In conclusion, TCR-based MRD quantification appears to be a suitable tool for monitoring and guiding treatment interventions in T-PLL. The MRD responses to immune modulation observed here provide first molecular evidence for GvL activity in T-PLL which, however, may be often only transient and reliant on a poly-/oligoclonal rather than a monoclonal T-cell response.

Centrosome defects and genetic instability in malignant tumors.

Genetic instability is a common feature of many human cancers. This condition is frequently characterized by an abnormal number of chromosomes, although little is known about the mechanism that generates this altered genetic state. One possibility is that chromosomes are missegregated during mitosis due to the assembly of dysfunctional mitotic spindles. Because centrosomes are involved in spindle assembly, they could contribute to chromosome missegregation through the organization of aberrant spindles. As an initial test of this idea, we examined malignant tumors for centrosome abnormalities using antibodies to the centrosome protein pericentrin. We found that centrosomes in nearly all tumors and tumor-derived cell lines were atypical in shape, size, and composition and were often present in multiple copies. In addition, virtually all pericentrin-staining structures in tumor cells nucleated microtubules, and they participated in formation of disorganized mitotic spindles, upon which chromosomes were missegregated. All tumor cell lines had both centrosome defects and abnormal chromosome numbers, whereas neither was observed in nontumor cells. These results indicate that centrosome defects are a common feature of malignant tumors and suggest that they may contribute to genetic instability in cancer.

Natural 30 base pair and 69 base pair deletion variants of the LMP1 oncogene do stimulate NF-kappaB-mediated transcription.

An increasing number of reports shows a link between the Epstein-Barr virus (EBV) and lymphoid neoplasia. The latent membrane protein 1 (LMP1) is likely to play a determinant role in this process since this EBV encoded protein has oncogenic properties and is usually expressed in EBV-associated lymphoproliferative diseases (LPD), except Burkitt's lymphoma. We previously identified in LPD patients mutational hot spots and a 30 bp or 69 bp deletion in the LMP1 gene region coding for the C-terminal domain. These deletions are located in an area shown to be important for the activation of the transcription factor NF-kappaB. These findings lead us to test whether these natural deletion variants may have a functional effect. We measured the stimulation of their activity using a luciferase reporter plasmid containing NF-kappaB responsive elements. We tested the NF-kappaB inducing activity of four naturally occurring LMP1 deletion variants. Our results show that these deletion variants activate NF-kappaB to the same level as the wild-type form, indicating that the crucial residues for NF-kappaB activation are conserved among the variants isolated and lie within the last 32 amino acids of the C-terminal domain of the LMP1 oncogene.

Expression of the LMP1 oncoprotein in the EBV negative Hodgkin's disease cell line L-428 is associated with Reed-Sternberg cell morphology.

The latent membrane protein 1 (LMP1) oncogene is one of the major proteins synthesized by the Epstein-Barr virus (EBV) and is expressed in Reed-Sternberg (RS) cells of EBV-associated Hodgkin's disease (HD). We have studied the effect of this oncoprotein on the formation of RS cells in the EBV-negative HD cell lines L-428 and KM-H2 as well as on the formation of multinucleated cells in the mononuclear human embryonic kidney cell line 293. LMP1 prototype (B95-8) and its naturally occurring carboxy terminal 30 base pair deletion variant LMP1-del were transfected into the cell lines and cytocentrifuge preparations were analysed after 24, 48, 72, 144, 216, and 240 h. While no oncoprotein expression was seen in the KM-H2 cell lines, expression of LMP1 and LMP1-del was observed in the L-428 and 293 cell lines. In the HD cell line L-428 oncoprotein expression was infrequent but when observed was very strong and preferentially associated with multinucleated RS cell morphology (71% of LMP1 positive cells). This is in contrast with the untransfected or transfected but not expressing cells where intermediate mononuclear elements predominated over multinucleated RS cells (< 3%). Frequent oncoprotein expression was observed in the 293 cells and again was associated with multinuclearity. These LMP1 expressing 293 giant cells showed strong expression of ICAM-1(CD54), not detectable in the untransfected cells. In the LMP1-del transfectants giant cells with more than four nuclei were frequently observed. However, giant cells were much less frequent in 293 cells transfected with the amino terminal deletion variant of LMP1 or the lytic form of LMP1, known to induce low NF-kappa B activation compared to the LMP1 prototype. Therefore, LMP1 mediated NF-kappa B activation appears to be involved in polycaria formation. The strong association of LMP1 expression with multinuclearity in a genetically unstable condition -the L-428 and 293 cells show multiple chromosomal abnormalities-suggests that this oncoprotein including its naturally occurring carboxy terminal deletion variant promote the formation of multinuclear cells, in particular of RS cells in EBV-associated HD.

Mutational hot spots within the carboxy terminal region of the LMP1 oncogene of Epstein-Barr virus are frequent in lymphoproliferative disorders.

We have recently identified in Epstein-Barr virus (EBV) positive Hodgkin's disease (HD) a variant of the latent membrane protein 1 (LMP1) oncogene characterized by four point mutations and a 30 base pair deletion. These findings led us to test whether such mutants were also present in other lymphoproliferative disorders (LPD). We analysed 98 EBV DNA positive cases (67 LPD, 15 benign conditions, 16 lymphoblastoid cell lines) by PCR for deletions within the LMP1 gene. DNA sequencing of the region coding for the carboxy terminal protein domain was performed on 24 cases. In 13 cases the same combination of 4 point mutations at positions 168,320, 168,308, 168,295 and 168,225 was identified. Of these cases, 12 had an additional point mutation at position 168,357 and eight at position 168,355, and nine had a 30 base pair deletion including nucleotides 168,285 to 168,256. These deletion mutants were identified in HD, angioimmunoblastic lymphadenopathy, B-immunoblastic lymphoma, peripheral T-cell lymphoma, and two lymphoblastoid cell lines. Our findings reveal a high frequency of non-random point mutations at preferential sites within the 3' (carboxy terminal) region of the LMP1 oncogene. The association of these mutational hot spots with LPD suggests that they are involved in EBV related lymphomagenesis and that they define a clinically relevant EBV strain.

Latent membrane protein 1 associated signaling pathways are important in tumor cells of Epstein-Barr virus negative Hodgkin's disease.

The latent membrane protein 1 (LMP1) oncogene of Epstein-Barr virus (EBV) is selectively expressed in the Reed-Sternberg (RS) cells of EBV-associated Hodgkin's disease (HD). However, no differences in clinical presentation and course are found between EBV positive and EBV negative forms of HD suggesting a common pathogenetic mechanism. We have studied the LMP1 associated signaling pathways and their dominant negative inhibition in the myelomonocytic HD-MyZ and the B-lymphoid L-428 HD cell lines. In both EBV negative cell lines expression of LMP1 is associated with the formation of multinuclear RS cells. Dominant negative inhibition of NF-kappa B mediated signaling at the step of I kappa B-alpha phosphorylation results in increased cell death with only a few typical RS cells resistant to overexpression of the dominant negative inhibitor I kappa B-alpha-N delta 54. However, dominant negative inhibition of NF-kappa B mediated signaling at the early step of TRAF2 interaction results in the formation of multinuclear cells in both cell lines and, in addition, in clusters of small mononuclear cells in the HD-MyZ cell line. In HD-MyZ cells overexpression of the powerful JBD-inhibitor of the JNK signal transduction pathway is restricted to small cells and never observed in RS cells. These small cells undergo apoptosis as shown by the TUNEL technique. Apoptosis of small cells is still observed after co-transfection of JBD and LMP1 but in addition a few apoptotic HD-MyZ cells with large fused nuclear masses are identified suggesting that specific inhibition of JNK leads also to apoptosis of LMP1 induced RS cells. Thus, activation of the JNK signaling pathway is also important in the formation of Reed-Sternberg cells. Our findings are consistent with a model where all three LMP1 associated functions, i.e. NF-kappa B mediated transcription, TRAF2 dependent signaling, and c-Jun activation act as a common pathogenetic denominator of both EBV negative and EBV positive HD.

Genotypic and phenotypic evidence of T-cell leukemia in a patient successfully treated by interferon-alpha for typical hairy cell leukemia.

A 53-year-old man was diagnosed to have typical hairy cell leukemia. Immunophenotyping of frozen splenic tissue showed clonality of hairy cells for mu lambda, confirmed by the corresponding immunoglobulin gene rearrangements. The patient was successfully treated with interferon-alpha (IF-alpha). In the fifth year of treatment with IF-alpha the morphology of peripheral blood mononuclear cells (PBMC) and of bone marrow infiltration changed with the appearance of numerous small to intermediate shaped lymphocytes of a T-helper phenotype. Frank leukemia, resistant to IF-alpha treatment and ultimately aggressive chemotherapy, developed. Emergence of this second clonal disease was confirmed by rearrangement studies performed on PBMC; rearrangements of both alleles of the TCR beta were identified, whereas the JH and lambda IVS genes were in germline configuration. The outgrowth of a second, malignant T-cell clone paralleled by the disappearance (down-regulation?) of the initial B-cell clone while under cytokine treatment is consistent with the possibility that IF-alpha favoured the emergence of this second clone.

A deletion mutant of the LMP1 oncogene of Epstein-Barr virus is associated with evolution of angioimmunoblastic lymphadenopathy into B immunoblastic lymphoma.

The latent membrane protein 1 (LMP1) oncogene is one of the major proteins synthesized by the Epstein-Barr virus (EBV). It is expressed in Reed-Sternberg cells of Hodgkin's disease (HD), tumor cells of nasopharyngeal carcinoma (NPC), and immunoblasts of angioimmunoblastic lymphadenopathy (AILD). A particular LMP1 deletion mutant was recently identified in NPC and clinically and histologically aggressive HD. We studied two patients with AILD that subsequently progressed into immunoblastic lymphoma (IBL) in order to investigate whether the LMP1 deletion mutant was implicated in progression of AILD into IBL. Immunohistology and in situ hybridization were performed on diagnostic biopsies. DNA extracted from fresh frozen material was used for rearrangement studies and polymerase chain reaction (PCR) based amplification and sequencing of portions of the LMP1 gene. Immunohistochemistry revealed B cell origin of both cases of IBL. In the first patient clonal rearrangement of the immunoglobulin heavy-chain gene was present in IBL but not in AILD. In this patient, scattered immunoblasts of AILD and numerous tumor cells of B-IBL were shown to contain EBV transcripts (EBER1) and to express LMP1. Sequence analysis of the LMP1 gene from AILD and IBL in the first, and from IBL in the second patient, revealed identical deletions and point mutations. This LMP1 deletion mutant is identical to those which have been reported in HD and NPC. Its association with evolution of AILD into B-IBL, aggressive HD and NPC, suggests that this particular mutant is more widespread than originally thought and is clinically relevant.

Molecular analysis of the LMP (latent membrane protein) oncogene in Hodgkin's disease.

Molecular analysis of the LMP (latent membrane protein) oncogene was performed in 21 Epstein-Barr virus (EBV) positive cases of Hodgkin's disease (HD) with proven LMP gene expression. In each case, viral DNA of the LMP gene was assessed for polymorphism (deletions, insertions, mutations) by polymerase chain reaction (PCR) amplification with selected primers. Specificity of the amplified targets was proven by internal oligonucleotide hybridisation and nested primer PCR. Homogeneity of the 5' LMP gene region coding for the amino terminal, transmembrane, and short extracytoplasmic domains of the protein was identified in all cases. However, deletions or insertions of small DNA sequences within the coding region for the intracytoplasmic LMP domain were observed in about 20% of cases. In one of them, a 30-base-pair deletion was precisely localized by DNA sequencing. A particularly high frequency of DNA polymorphism (30% of cases) was found in the 3' untranslated LMP region. However, when analysing the LMP gene in seven benign conditions, no DNA polymorphism was found. These data suggest conservation of oncogenic LMP regions coding for the protein domains known to be associated with transforming capacities and immunogenic functions. They also show a considerable genomic heterogeneity of the coding region for the intracytoplasmic domain and the 3' untranslated mRNA region. This LMP DNA polymorphism identified within a localized (Swiss) population suffering from HD is unexpected. Its eventual clinical significance remains to be determined.

Molecular and functional analysis of the Epstein-Barr virus LMP1 oncogene promoter in lymphoproliferative diseases.

The expression of the Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) oncogene is tightly regulated by viral and cellular factors. LMP1 is present in the majority of nasopharyngeal carcinoma tumor cells and in Reed-Sternberg cells from Hodgkin's lymphoma, in which the only EBV nuclear antigen detected is EBNA1. The aim of this study was to test whether mutations affecting LMP1 gene expression were present in lymphoproliferative disorders, and, if so, whether their presence correlated with the clinical course of the disease. For this purpose we characterized the LMP1 promoter region from seven cases including two patients with aggressive Hodgkin's disease and two with atypical lymphoproliferative syndromes. Our results show that the sequences -298 to +29 relative to the transcription start site diverged up to 9.3% when compared with the prototype EBV strain B95-8. The cAMP responsive-like element (CRE), located at positions -37 to -44, was found to be mutated in 3 of the 7 cases. Functional analysis of transfected human embryonic kidney 293 cells using the firefly luciferase reporter gene revealed that mutations within the CRE site led to a 70% mean decrease in reporter activity. Our analysis indicates that in lymphoproliferative disorders, naturally occurring LMP1 variants that exhibit weak promoter activity are still associated with clinically progressive disease.

Relationship between the duration of paralysis and bone structure: a pQCT study of spinal cord injured individuals.

The aim of the present study was to describe bone loss of the separate compartments of trabecular and cortical bone, as well as changes in bone geometry of a large number of spinal cord injured (SCI) individuals. Eighty-nine motor complete spinal cord injured men (24 tetraplegics and 65 paraplegics) with a duration of paralysis of between 2 months and 50 years were included in the study. Distal epiphyses and midshafts of the femur, tibia, and radius were measured by peripheral quantitative computed tomography. The same measurements were performed in a reference group of 21 healthy able-bodied men of the same age range. In the femur and tibia, bone mass, total and trabecular bone mineral density (BMDtot and BMDtrab, respectively) of the epiphyses, as well as bone mass and cortical cross-sectional area of the diaphyses, showed an exponential decrease with time after injury in the spinal cord injured subjects. The decreasing bone parameters reached new steady states after 3-8 years, depending on the parameter. Bone mass loss in the epiphyses was approximately 50% in the femur and 60% in the tibia, while the shafts lost only approximately 35% in the femur and 25% in the tibia. In the epiphyses, bone mass was lost by reducing BMD, while in the shaft bone mass was lost by reducing cortical wall thickness, a process achieved by endosteal resorption advancing at a rate of about 0.25 mm/year within the first 5-7 years after injury. Except for a slight transient decrease in cortical BMD of the femoral and tibial shaft during the first 5 years after the spinal cord lesion, cortical BMD of the spinal cord injured subjects was found to be at reference values. Bone parameters of the radial epiphysis in paraplegic subjects showed no deficits compared to the reference group. Furthermore, a trend for an increased radial shaft diameter suggests periosteal apposition as a consequence of increased loading of the arms.

Inflammatory pseudotumor of the liver. Evidence for follicular dendritic reticulum cell proliferation associated with clonal Epstein-Barr virus.

We describe an "inflammatory pseudotumor" of the liver that, which on detailed investigation, proved that the spindle-cell component of this lesion is derived from follicular dendritic reticulum cells (FDRC). This contention is supported by morphologic observations and by immunophenotype. The FDRC population contain Epstein-Barr virus (EBV). It is known that FDRC express the EBV receptor CD21. In this particular case, the FDRC contained clonal EBV genomes, EBV RNA (EBER) transcripts, and expressed EBV latent membrane protein (LMP1). DNA sequencing of PCR products showed three point mutations compared with the standard LMP1 sequence of the EBV strain B95-8. The findings in this case corroborate those of other investigators concerning the possible role of EBV in the development of some inflammatory pseudotumors, including the recent production of functionally active EBV-transformed FDRC-like cell lines. This association could prove instructive in delineating the histogenesis of these tumors and further assist in making prognostic and therapeutic decisions.

Molecular pathogenesis of Epstein-Barr virus associated posttransplant lymphomas: new insights through latent membrane protein 1 fingerprinting.

BACKGROUND: Fingerprint amino acid patterns within the carboxy terminus of the latent membrane protein (LMP1) oncoprotein of Epstein-Barr virus (EBV) allow individual strain identification at the molecular level. LMP1 is expressed in the tumor cells of EBV-associated posttransplant lymphomas (PTLs) and the LMP1 genome is also identified in lymphocytes of most donors of allogeneic bone marrow. Therefore, LMP1 genotyping in donor lymphocytes and PTL tumor cells, together with sex chromatin determination of tumor cells, allows to determine the origin of PTL tumor cells and the origin of individual EBV strains harboured by them. METHODS: We traced the origin of aggressive PTLs occurring in six patients after allogeneic T cell-depleted stem cell transplantation (allo-SCT). DNA was extracted from donor lymphocytes and PTLs of recipients and amplified with LMP1-specific primers in each case. A comparative sequence analysis of the fingerprint LMP1 region identified in donor lymphocytes and lymphoma was performed. RESULTS: One lymphoma of donor origin occurred after highly selected CD34+ PBSCT and contained the same LMP1 genotype as the donor lymphocytes. Three lymphomas of recipient origin had deletions within the carboxy terminus of LMP1, not identified in the donor strains. All lymphomas occurred in the setting of allo-SCT and had a rapid clinical course. CONCLUSIONS: These results show that highly selected CD34+ PBSCT does not protect against transfer of EBV positive founder cells of donor type PTL and that, after allo-SCT, recipient type PTLs are not uncommon. Outgrowth of recipient type lymphoma may be favoured by LMP1 deletion variant strains present in recipient lymphocytes.

Selective outgrowth of a posttransplant B-immunoblastic lymphoma expressing a latent membrane protein-1 deletion variant.

BACKGROUND: Posttransplant lymphoproliferative disorders are generally associated with Epstein-Barr virus (EBV) and are of B cell origin. We report the case of a B-immunoblastic lymphoma that developed in a pretransplantation EBV-seronegative woman 4 months after kidney transplant from her HLA-haploidentical brother. The patient successfully underwent immunotoxin therapy for lymphoma and has been in remission for 36 months. METHODS: Latent EBV genomes were identified by polymerase chain reaction, and the purified amplification products were directly sequenced with [35S]dATP. RESULTS: Molecular analysis of the latent membrane protein (LMP)1 oncogene of EBV, which was expressed in most tumor cells, revealed a 30-base pair deletion. No wild-type LMP1 sequences were found. Analysis of peripheral blood mononuclear cells from the EBV-seropositive donor showed the presence of both the LMP1 deletion variant and the wild-type sequence. The LMP1 deletion variant and the wild-type sequence were also identified within peripheral blood mononuclear cells of the EBV-seroconverted kidney recipient 20 months after lymphoma therapy. CONCLUSION: This pattern is consistent with a natural growth advantage of B cells expressing the LMP1 deletion variant in the immunocompromised host.

Deletion variants within the NF-kappaB activation domain of the LMP1 oncogene in acquired immunodeficiency syndrome-related large cell lymphomas, in prelymphomas and atypical lymphoproliferations.

The latent membrane protein 1 (LMP1) oncogene of Epstein Barr virus (EBV) is expressed in tumor cells of acquired immunodeficiency syndrome (AIDS) related lymphomas, HIV-negative, EBV-associated malignant lymphoproliferations, nasopharyngeal carcinoma, as well as in reactive immunoblasts of infectious mononucleosis. Naturally occurring LMP1 deletion variants (LMP1-del), characterized by clustered mutations and a distinct 30 base pair deletion within the carboxy terminal domain of LMP1, essential for maximal NF-kappaB stimulation, have been identified in the same conditions. These variants prevail in AIDS-related lymphomas, and are associated with clinically aggressive behaviour in HIV-negative lymphomas, and are frequent in prelymphomatous and reactive states. Functional studies showing a growth advantage of cells infected by these variants may explain the accumulation of LMP1-del in these entities. In the carboxy terminal NF-kappaB activation domain of LMP1, evidence of a hypervariable region close to the highly conserved 23 outermost amino acids essential for malignant transformation, may reflect the natural selection of growth promoting variants involved in signalling pathways. The prevalence of the same mutational pattern in AIDS-related lymphoma as well as in hyperplastic reactive states and prelymphomas supports the hypothesis that these variants confer a growth advantage manifested under impaired cellular immunity.

Expression of human recombination activating genes (RAG-1 and RAG-2) in lymphoma.

Two recently discovered genes, the recombination activating genes 1 and 2 (RAG-1 and RAG-2), are necessary to perform variable (V), diversity (D), and joining (J) recombination. They synergistically activate VDJ recombination to generate immunocompetent lymphocytes. Disruption of either gene results in a maturation arrest at a very early B and T cell progenitor stage. Expression and downregulation of RAG's are closely associated with interleukin 7, sIgM and TCR-CD3 complex, respectively. Assessment of RAG mRNA expression is a valuable marker in identifying the genotypic maturation status of leukemias and lymphomas. Persistent RAG expression in otherwise mature lymphoid proliferations may explain puzzling biological and clinical observations such as multiple rearrangements in lymphomas with a mature phenotype. Lack of RAG expression in Hodgkin's disease with abundant Reed-Sternberg cells is consistent with a mature phenotype of the latter. Availability of a anti-RAG-1 monoclonal antibody in the near future will facilitate RAG analysis of lymphomas.

Significance of the detection of Epstein-Barr virus DNA in lymph nodes in patients with Hodgkin's disease.

Epstein-Barr virus (EBV) DNA is frequently identified in benign and malignant lymphoproliferative conditions. As shown by in situ hybridization studies viral DNA is localized within malignant cells as well as benign lymphocytes. Clonal and nonclonal EBV genomes are present in Hodgkin's disease (HD), lymphomas of the immunocompromised host and reactive lymph node hyperplasia. Lytic infection with formation of linear genomes is observed in the same conditions but appears to be infrequent in HD as shown by quantitation of mRNA coding for viral capsid antigen. Expression of the oncogene LMP (latent membrane protein) is seen in Sternberg-Reed (SR) cells and immunoblasts of AIDS-related lymphoma and infectious mononucleosis (IM). In HD, the region of the BNLF1 oncogene coding for the amino terminal and transmembrane domains (associated with oncogenic function) of LMP appears to be homogeneous whereas the region coding for the intracytoplasmic (carboxy terminal) domain of LMP is heterogeneous. Cytological similarities between SR cells and immunoblasts of IM and AIDS-related lymphomas are consistent with the hypothesis that the BNLF1 oncogene is one possible inducer of morphological features of SR cells. Whether chromosomal integration of EBV DNA is an important factor in activation of such a transforming activity remains to be elucidated. EBV DNA positive and negative HD cases with numerous SR cells lack significant mRNA expression of the two recombinase activating genes (RAG-1 and RAG-2). Therefore the SR cells appear to be derived from lymphocytes beyond the pre-B-cell or common thymocyte stage which may or may not subsequently become infected by EBV.

Synthesis, biodistribution and renal handling of various chelate-somatostatin conjugates with metabolizable linking groups.

A series of DTPA-octreotide conjugates, with various linking groups, were synthesised to investigate the effect of different metabolizable linkers on the renal retention of radioactivity. All these newly synthesised octreotide conjugates retained the high binding affinity of octreotide for the somatostatin (SRIF) receptors either when unlabeled or radiolabeled with 111In. Some of the metabolizable linkers were rapidly degraded in vitro when incubated with a kidney homogenate. However, in vivo, all these conjugates displayed a significantly lower uptake in SRIF receptor-positive tissue compared to two conjugates with short, stable linkers. Additionally, the compounds with a potentially metabolizable linker had a higher whole-body retention of activity as opposed to the three metabolically stable compounds. Several of the linkers gave evidence of cleavage while in circulation in the blood, and it is probable that the lower tumour accumulation of most of the compounds tested was low due to the high enzymatic nature of the exocrine pancreatic tumour model used. In short, no increase in the tumour-to-kidney ratio was achieved with the analogues containing a metabolizable linker. The highest target-to-nontarget tissue ratios were obtained for the DTPA-octreotide conjugates that had short, metabolically stable linkers.

Molecular analysis of critical sequences within the EBNA-2 type 1 gene from Epstein-Barr virus isolates from patients with infectious mononucleosis, tonsillar hyperplasia, and HIV infection.

EBNA-2 is the first protein to be detected after infection of primary B lymphocytes by Epstein-Barr virus (EBV) and plays an essential role as transcriptional activator in EBV-induced lymphocyte transformation. We analysed by PCR and sequencing regions of the EBNA-2 type 1 gene from isolates from 13 children with infectious mononucleosis (IM), 6 children with tonsillar hyperplasia (TH), and 9 patients with HIV infection followed longitudinally. We found in all three groups of patients frequent non-silent point mutations at positions 48990, 48991, 49021, 49057, 49083, 49089, 49091, 49113, 49119, 49140, 49156, and a triplet insertion at position 49136. While 4 out of 13 samples from patients with IM showed a mosaic pattern suggesting co-existence of more than 1 substrain of EBNA-2 type 1, none of the samples from TH showed this pattern consistent with substrain selection during clinical latency. No sequence changes were noted over time in samples derived from patients with HIV infection. We conclude that in analogy to the coexistence of several subtypes of EBNA-1 in healthy EBV carriers, samples from IM can harbor more than one subtype of the EBNA-2 type 1 gene.

Comparison of three methods used to obtain a neutral plaster foot impression.

The purpose of our study was to compare the forefoot-to-hindfoot angles obtained from three methods used to obtain a neutral plaster impression of the foot. The three methods were 1) supine nonweight-bearing (S), 2) prone nonweight-bearing (P), and 3) sitting semiweight-bearing (SW). We obtained foot casts from both feet of 11 female subjects for each of the three methods and used a manual goniometer to measure the forefoot-to-hindfoot angle for each pair of casts. The F ratios were significant for the variables left-right foot (p less than .0001) and impression method (p less than .001) using a within-subject two-factor analysis of variance. The impression methods S and P were found to be significantly different from SW, but not significantly different from each other, using a Tukey's post hoc comparison. The results indicate that the same forefoot-to-hindfoot alignment can be obtained using either the S or P method but not with the SW method.