Within- and between-therapist agreement on personalized parameters for robot-assisted gait therapy: the challenge of adjusting robotic assistance.

BACKGROUND: Stationary robotic gait trainers usually allow for adjustment of training parameters, including gait speed, body weight support and robotic assistance, to personalize therapy. Consequently, therapists personalize parameter settings to pursue a relevant therapy goal for each patient. Previous work has shown that the choice of parameters influences the behavior of patients. At the same time, randomized clinical trials usually do not report the applied settings and do not consider them in the interpretation of their results. The choice of adequate parameter settings therefore remains one of the major challenges that therapists face in everyday clinical practice. For therapy to be most effective, personalization should ideally result in repeatable parameter settings for repeatable therapy situations, irrespective of the therapist who adjusts the parameters. This has not yet been investigated. Therefore, the aim of the present study was to investigate the agreement of parameter settings from session to session within a therapist and between two different therapists in children and adolescents undergoing robot-assisted gait training. METHODS AND RESULTS: Fourteen patients walked in the robotic gait trainer Lokomat on 2 days. Two therapists from a pool of 5 therapists independently personalized gait speed, bodyweight support and robotic assistance for a moderately and a vigorously intensive therapy task. There was a very high agreement within and between therapists for the parameters gait speed and bodyweight support, but a substantially lower agreement for robotic assistance. CONCLUSION: These findings imply that therapists perform consistently at setting parameters that have a very clear and visible clinical effect (e.g. walking speed and bodyweight support). However, they have more difficulties with robotic assistance, which has a more ambiguous effect because patients may respond differently to changes. Future work should therefore focus on better understanding patient reactions to changes in robotic assistance and especially on how instructions can be employed to steer these reactions. To improve the agreement, we propose that therapists link their choice of robotic assistance to the individual therapy goals of the patients and closely guide the patients during walking with instructions.

Image-plate sensitivity to x rays at 2 to 60 keV.

The sensitivity of Fuji SR and MS image plates (IPs) used in x-ray spectrometers on OMEGA and the National Ignition Facility has been measured using two techniques. A set of radioisotopes has been used to constrain image-plate sensitivity between 6 and 60 keV, while a Manson source has been used to expose image plates to x rays at energies between 1.5 and 8 keV. These data have shown variation in sensitivity on the order of 5% for a given IP type and scanner settings. The radioisotope technique has also been used to assess IP fading properties for MS-type plates over long times. IP sensitivity as a function of scanner settings and pixel size has been systematically examined, showing variations of up to a factor of 2 depending on the IP type. Cross-calibration of IP scanners at different facilities is necessary to produce a consistent absolute sensitivity curve spanning the energy range of 2-60 keV.

Prospective comparison of diffusion-weighted MRI and dynamic Gd-EOB-DTPA-enhanced MRI for detection and staging of hepatic fibrosis in primary sclerosing cholangitis.

PURPOSE: To assess the diagnostic value of multiparametric magnetic resonance imaging (MRI) including dynamic Gd-EOB-DTPA-enhanced (DCE) and diffusion-weighted (DW) imaging for diagnosis and staging of hepatic fibrosis in primary sclerosing cholangitis (PSC) using transient elastography as a standard reference. MATERIAL AND METHODS: Multiparametric MRI was prospectively performed on a 3.0-Tesla scanner in 47 patients (age 43.9±14.3 years). Transient elastography derived liver stiffness measurements (LSM), DCE-MRI derived parameters (hepatocellular uptake rate (Ki), arterial (Fa), portal venous (Fv) and total (Ft) blood flow, mean transit time (MTT), and extracellular volume (Ve)) and the apparent diffusion coefficient (ADC) were calculated. Correlation and univariate analysis of variance with post hoc pairwise comparison were applied to test for differences between LSM derived fibrosis stages (F0/F1, F2/3, F4). ROC curve analysis was used as a performance measure. RESULTS: Both ADC and Ki correlated significantly with LSM (r= -0.614; p<0.001 and r= -0.368; p=0.01). The ADC significantly discriminated fibrosis stages F0/1 from F2/3 and F4 (p<0.001). Discrimination of F0/1 from F2/3 and F4 reached a sensitivity/specificity of 0.917/0.821 and 0.8/0.929, respectively. Despite significant inter-subject effect for classification of fibrosis stages, post hoc pairwise comparison was not significant for Ki (p>0.096 for F0/1 from F2/3 and F4). LSM, ADC and Ki were significantly associated with serum-based liver functional tests, disease duration and spleen volume. CONCLUSION: DW-MRI provides a higher diagnostic performance for detection of hepatic fibrosis and cirrhosis in PSC patients in comparison to Gd-EOB-DTPA-enhanced DCE-MRI. KEY POINTS: • Both ADC and hepatocellular uptake rate (Ki) correlate significantly with liver stiffness (r= -0.614; p<0.001 and r= -0.368; p=0.01). • The DCE-imaging derived quantitative parameter hepatocellular uptake rate (Ki) fails to discriminate pairwise intergroup differences of hepatic fibrosis (p>0.09). • DWI is preferable to DCE-imaging for discrimination of fibrosis stages F0/1 to F2/3 (p<0.001) and F4 (p<0.001).

The immune response to sporadic colorectal cancer in a novel mouse model.

Current mouse models do not reflect the sporadic nature of colon cancer and do not allow the analysis of antitumor immune response because of the lack of known tumor-specific antigens. Two transgenic mouse models with spontaneous tumor development were generated, directing the expression of SV40T antigen (Tag) either constitutively (Vil-Cre × LoxP-Tag-transgenic mice) or stochastically (Vil-Cre-ER(T2) × LoxP-Tag-transgenic mice) into the putative stem cell region of the crypt of Lieberkühn. Tumor development and antitumor immune response were monitored. Vil-Cre × LoxP-Tag mice developed multiple adenocarcinomas of the small intestine and colon at an average age of 6 months. During the tumor development, Tag-specific immunoglobulin G (IgG) antibodies were induced in half of the mice, although they had developed neonatal cytotoxic T lymphocyte (CTL) tolerance. This model shows similarity to hereditary colon cancer but not to the sporadic tumor development. Therefore, the conditional Vil-Cre-ER(T2) × LoxP-Tag mice were established, in which expression of the dormant Tag was induced by stochastic, tissue-specific activation of Cre recombinase. These mice spontaneously developed highly invasive, metastasizing colon carcinomas at an average age of 20 months. Colon carcinomas expressed epithelial and/or neuroendocrine markers depending on the grade of differentiation. Young Vil-Cre-ER(T2) × LoxP-Tag mice had retained CTL responses against epitope IV of Tag. The tumors induced strong anti-Tag IgG responses. We report, for the first time, a mouse model based on stochastic, tissue-specific activation of a dormant oncogene in the colon allowing the analysis of antitumor immune response against primary colorectal cancer.

A randomized, double-blind trial to compare shock wave frequencies of 60 and 120 shocks per minute for upper ureteral stones.

PURPOSE: Recent evidence demonstrates that decreasing shock wave frequency from the previous standard of 120 to 60 shocks per minute results in improved fragmentation of stones located within the renal collecting system. We report the first randomized trial to our knowledge to examine the effect of a slower shock wave frequency for shock wave lithotripsy on stones located in the proximal ureter. MATERIALS AND METHODS: A total of 163 patients with a previously untreated radiopaque calculus in the upper ureter measuring at least 5 mm underwent stratified block randomization according to stone size, and shock wave lithotripsy at 60 or 120 shocks per minute. Stone-free status at 3 months was confirmed with noncontrast computerized tomography or a plain abdominal x-ray and ultrasound study. RESULTS: Of the patients 77 were randomized to 60 shocks per minute and 86 were randomized to 120 shocks per minute. The groups were similar in gender, age, body mass index and initial stone area. At 3 months the 60 shocks per minute group had a higher overall stone-free rate (64.9% vs 48.8%, p = 0.039). Significantly fewer shocks were administered to patients treated at 60 shocks per minute (mean 2,680 vs 2,940, p <0.001). However, mean treatment times were longer (44.3 vs 24.5 minutes, p <0.001). Patients treated with 60 shocks per minute required fewer auxiliary procedures (29.9% vs 45.4%) (p = 0.031). CONCLUSIONS: Decreasing the rate of shock wave administration from 120 to 60 shocks per minute results in improved stone-free rates. A slower treatment rate of proximal ureteral stones reduces the need for additional shock wave lithotripsy or more invasive treatments to render patients stone-free, without any increase in morbidity, and with an acceptable increase in treatment time.

A true singles list-mode data acquisition system for a small animal PET scanner with independent crystal readout.

We present a unique data acquisition system designed to read out signals from the MADPET-II small animal LSO-APD PET tomograph. The scanner consists of 36 independent detector modules arranged in a dual-radial layer ring (phi 71 mm). Each module contains a 4 x 8 array of optically isolated, 2 x 2 mm LSO crystals, coupled one-to-one to a 32 channel APD. To take full advantage of the detector geometry, signals from each crystal are individually processed without any data reduction. This is realized using custom designed mixed-signal ASICs for analogue signal processing, and FPGAs to control the digitization of analogue signals and subsequent multiplexing. Analogue to digital converters (ADCs) digitize the signal peak height, time to digital converters (TDCs) time stamp each event relative to a system clock and two 32 bit words containing the energy, time and position information for each singles event are multiplexed through three FIFO stages before being written to disk via gigabit Ethernet. Every singles event is processed and stored in list-mode format, and coincidences are sorted post-acquisition in software. The 1152 channel data acquisition system was designed to be able to handle sustained data rates of up to 11 520 000 cps without loss (10 000 cps/channel). The timing resolution of the TDC was measured to be 1 ns FWHM. In addition to describing the data acquisition system, performance measurements made using a 128-channel detector prototype will be presented.

Laboratory studies of the effects of reduced prey choice caused by Bt plants on a predatory insect.

Crops transformed to express Bacillus thuringiensis (Bt) toxins can cause close to 100% mortality of certain target pest species. This study assessed the effect of target pest reduction on the predatory insect Chrysoperla carnea (Stephens) in the presence of alternative prey. Numbers of lacewings recovered from Bt oilseed rape (cultivar Oscar, event O52) did not differ significantly from numbers of lacewings recovered from conventional oilseed rape in cage experiments with the target pest Plutella xylostella (Linnaeus) and the non-target pest Myzus persicae (Sulzer) when aphid densities were high. However, significantly fewer lacewings were recovered from Bt plants as aphid densities were lowered. Lacewing weights were not affected by plant type.

Molecular interactions of the type 1 human immunodeficiency virus transregulatory protein Tat with N-methyl-d-aspartate receptor subunits.

We investigated the effect of type 1 human immunodeficiency virus (HIV-1) regulatory protein Tat on N-methyl-d-aspartate (NMDA) receptors expressed in Xenopus oocytes by voltage-clamp recording and its role in NMDA-mediated neurotoxicity using cultured rat hippocampal neurons. Tat (0.01-1muM) potentiated NMDA-induced currents of recombinant NMDA receptors. However, in the presence of Zn(2+), the potentiating effect of Tat was much more pronounced, indicating an additional Zn(2+)-related effect on NMDA receptors. Consistently, Tat potentiated currents of the particularly Zn(2+)-sensitive NR1/NR2A NMDA receptor with a higher efficacy, whereas currents from a Zn(2+)-insensitive mutant were only marginally augmented. In addition, chemical-modified Tat, deficient for metal binding, did not reverse Zn(2+)-mediated inhibition of NMDA responses, demonstrating that Tat disinhibits NMDA receptors from Zn(2+)-mediated antagonism by complexing the cation. We therefore investigated the interplay of Tat and Zn(2+) in NMDA-mediated neurotoxicity using cultures of rat hippocampal neurons. Zn(2+) exhibited a prominent rescuing effect when added together with the excitotoxicant NMDA, which could be reverted by the Zn(2+)-chelator tricine. Similar to tricine, Tat enhanced NMDA-mediated neurotoxicity in the presence of neuroprotective Zn(2+) concentrations. Double-staining with antibodies against Tat and the NR1 subunit of the NMDA receptor revealed partial colocalization of the immunoreactivities in membrane patches of hippocampal neurons, supporting the idea of a direct interplay between Tat and glutamatergic transmission. We therefore propose that release of Zn(2+)-mediated inhibition of NMDA receptors by HIV-1 Tat contributes to the neurotoxic effect of glutamate and may participate in the pathogenesis of AIDS-associated dementia.

Tumor rejection by disturbing tumor stroma cell interactions.

The stroma of solid tumors is a complex network of different cell types. We analyzed stroma cell interactions in two tumor models during cyclophosphamide (Cy)-induced tumor rejection. In growing tumors, tumor infiltrating macrophages (TIMs) produced interleukin (IL)-10. Beginning 6 h after Cy-treatment T cells in the tumor were inactivated and TIMs switched to interferon (IFN)-gamma production. Both, IL-10 production before and IFN-gamma production after Cy-treatment by TIMs required T cells. With the same kinetics as TIMs started to produce IFN-gamma the tumor vasculature was destroyed which required IFN-gamma receptor expression on host but not tumor cells. These events preceded hemorrhagic necrosis and residual tumor cell elimination by T cells. Together, T cells regulate the function of TIMs and tumor rejection can be induced by disturbing the stroma network.

Generation of tumor-associated cytotoxic T lymphocytes requires interleukin 4 from CD8(+) T cells.

Activation of tumor-associated CD8(+) cytotoxic T lymphocytes (CTLs) often requires antigen representation, e.g., by dendritic cells (DCs), and CD4(+) T cell help. Previously, we showed that CTL-mediated tumor immunity required interleukin 4 (IL-4) during the immunization but not effector phase. To determine the source and target cells of IL-4, we performed adoptive T cell transfers using CD4(+) and CD8(+) T cells from IL-4(-/-) and IL-4R(-/-) mice and analyzed CTL generation. Even though necessary for CTL generation, CD4(+) T cells did not need to express IL-4 or IL-4R. Surprisingly, CTL generation required IL-4 but not IL-4R expression by CD8(+) T cells. As IL-4 (a) was expressed by naive CD8(+) T cells within 24 h after antigen encounter, (b) IL-4 induced DC maturation, and (c) CTL development was impaired in T cell-reconstituted IL-4R(-/-) mice, CD8(+) T cell-derived IL-4 appears to act on DCs. We conclude that CD4(+) and CD8(+) T cells provide different signals for DC activation during CTL generation.

Mice overexpressing genes from the 22q11 region deleted in velo-cardio-facial syndrome/DiGeorge syndrome have middle and inner ear defects.

Velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS) is a congenital anomaly disorder associated with hemizygous 22q11 deletions. We previously showed that bacterial artificial chromosome (BAC) transgenic mice overexpressing four transgenes, PNUTL1, (CDCrel-1), GP1B beta, TBX1 and WDR14, had reduced viability, cardiovascular malformations and thymus gland hypoplasia. Since these are hallmark features of VCFS/DGS, we analyzed the mice for additional anomalies. We found that the mice have important defects in the middle and inner ear that are directly relevant to the disorder. The most striking defect was the presence of chronic otitis media, a common finding in VCFS/DGS patients. In addition, the mice had a hyperactive circling behavior and sensorineural hearing loss. This was associated with middle and inner ear malformations, analogous to Mondini dysplasia in humans reported to occur in VCFS/DGS patients. We propose that overexpression of one or more of the transgenes is responsible for the etiology of the ear defects in the mice. Based upon its pattern of expression in the ear and functional studies of the gene, TbX1 likely plays a central role. Haploinsufficiency of TBX1 may be responsible for ear disorders in VCFS/DGS patients.

Adoptive tumor therapy with T lymphocytes enriched through an IFN-gamma capture assay.

Successful adoptive T-cell therapy has been demonstrated in viral disease and selected forms of cancer. However, it is limited by the difficulty to efficiently isolate and amplify autologous tumor-reactive T-cell clones. Tetramers of major histocompatibility complex (MHC) class I and peptide have facilitated the characterization of CD8+ T cells specific for tumor-associated antigens. However, for adoptive T-cell therapy, MHC-tetramers have limitations: they require knowledge of tumor antigens, which is often not available; they select T cells with a single specificity, thereby posing risk for selection of tumor escape variants; they do not select for function, so that T cells may be anergic when isolated from cancer patients; and they do not allow the isolation of CD4+ T cells that can be essential for tumor rejection. Because interferon (IFN)-gamma is essential for tumor rejection, we isolated live T cells based on their IFN-gamma production. IFN-gamma secreted by previously activated T cells is retained on the cell surface, allowing their specific isolation and expansion. We show here that IFN-gamma+ but not IFN-gamma- T cells from tumor-immunized mice are cytolytic and mediate tumor rejection upon adoptive transfer. Importantly, tumor-specific T cells can be enriched from lymphocytes infiltrating human renal cell carcinoma by the IFN-gamma capture assay.

Population-scale laboratory studies of the effect of transgenic plants on nontarget insects.

Studies of the effects of insect-resistant transgenic plants on beneficial insects have, to date, concentrated mainly on either small-scale "worst case scenario" laboratory experiments or on field trials. We present a laboratory method using large population cages that represent an intermediate experimental scale, allowing the study of ecological and behavioural interactions between transgenic plants, pests and their natural enemies under more controlled conditions than is possible in the field. Previous studies have also concentrated on natural enemies of lepidopteran and coleopteran target pests. However, natural enemies of other pests, which are not controlled by the transgenic plants, are also potentially exposed to the transgene product when feeding on hosts. The reduction in the use of insecticides on transgenic crops could lead to increasing problems with such nontarget pests, normally controlled by sprays, especially if there are any negative effects of the transgenic plant on their natural enemies. This study tested two lines of insect-resistant transgenic oilseed rape (Brassica napus) for side-effects on the hymenopteran parasitoid Diaeretiella rapae and its aphid host, Myzus persicae. One transgenic line expressed the delta-endotoxin Cry1Ac from Bacillus thuringiensis (Bt) and a second expressed the proteinase inhibitor oryzacystatin I (OC-I) from rice. These transgenic plant lines were developed to provide resistance to lepidopteran and coleopteran pests, respectively. No detrimental effects of the transgenic oilseed rape lines on the ability of the parasitoid to control aphid populations were observed. Adult parasitoid emergence and sex ratio were also not consistently altered on the transgenic oilseed rape lines compared with the wild-type lines.

Acute and long-term humoral immunity following active immunization of rabbits with inactivated spores of various Encephalitozoon species.

Microsporidia of the genus Encephalitozoon are increasingly being reported as a cause of severe, often disseminated infections, mainly in patients with acquired immunodeficiency syndrome (AIDS). Immunological identification of each of the three recognized species (E. cuniculi, E. hellem, and E. intestinalis) requires the availability of specific immune sera. All sera available thus far have been generated by direct inoculation of rabbits with virulent microsporidian spores. This study demonstrates for the first time that subcutaneous immunization with inactivated spores of E. cuniculi, E. helleri, or E. intestinalis is capable of generating highly active rabbit hyperimmune sera to the homologous antigens, with maximal titers being 1:5,120, 1:1,280, and 1:2,560, respectively, as determined by the indirect immunofluorescence technique (IIF). Broad cross-reactivity of the rabbit antisera with all heterologous Encephalitozoon antigens was determined by IIF and immunogold electron microscopy; however, only the E. hellem immune serum strongly cross-reacted with spores of Enterocytozoon bieneusi. During the 35-month follow-up period the antibody titers to the homologous antigens declined to 1:640, 1:160, and 1:320, respectively. The observed decay curves for antibody titers against E. cuniculi, E. hellem, and E. intestinalis were fitted using mathematical modeling, resulting in a predicted duration for specific immune responses of about 7 years on average. Knowledge of the magnitude and duration of specific immune responses is a prerequisite for further evaluation of the concept of using inactivated microsporidian spores in the quest for vaccines against microsporidian infections.

Presentation by scanning electron microscopy of the life cycle of microsporidia of the genus Encephalitozoon.

This paper presents, for the first time, documentation by detailed scanning electron microscopy of the life cycle of microsporidia of the genus Encephalitozoon. Phase 1 is represented by the extracellular phase with mature spores liberated by the rupture of host cells. To infect new cells the spores have to discharge their polar filament. Spores with everted tubes show that these are helically coiled. When the polar tubules have started to penetrate into a host cell they are incomplete in length. The infection of a host cell can also be initiated by a phagocytic process of the extruded polar filament into an invagination channel of the host cell membrane. After the penetration process, the tube length is completed by polar tube protein which passes through the tube in the shape of swellings. A completely discharged polar tube with its tip is also shown. The end of a polar tube is normally hidden in the cytoplasm of the host cell. After completion of the tube length the transfer of the sporoplasm occurs and phase 2 starts. Phase 2 is the proliferative phase, or merogony, with the intracellular development of the parasite that cannot be documented by scanning electron microscopy. The subsequent intracellular phase 3, or sporogony, starts when the meronts transform into sporonts, documented as chain-like structures which subdivide into sporoblasts. The sporoblasts finally transform directly into spores which can be seen in their host cell, forming bubble-like swellings in the cell surface.

Chitinolytic activity in viable spores of Encephalitozoon species.

By employing 4-methylumbelliferyl-beta-D-NN',N"-triacetylchitotriose substrate in a semi quantitative assay, chitinolytic activity in viable spores of Encephalitozoon cuniculi and E. intestinalis was detected and dependence on reaction time, spore concentration, concentration of substrate and temperature were demonstrated. It was possible to block the chitinolytic activity by chitin hydrolysate. By incubation at 80 degrees C for 10 min or at 55 degrees C for 20 min the spores were loosing the chitinolytic activity. Incubation of the spores in trypsin reduced the chitinolytic activity. Cellulase activity could not be detected.

Surgical irrigation with pooled human immunoglobulin G to reduce post-operative spinal implant infection.

A multiple-site, nonlethal rabbit surgical model of spinal implant infection was used to assess the efficacy of a spinal wound lavage to reduce post-operative infection from methicillinresistant Staphylococcus aureus (MRSA). Multiple aqueous lavages of isotonic saline were compared to the same procedure using 1wt% pooled human immunoglobulin G (IgG) applied directly to the surgical implant sites. Visually observed clinically relevant signs of infection (e.g. , swelling, erythema, pus) were supported by bacterial enumeration from multiple biopsied tissue and bone sites post-mortem at 7 and 28 days post-challenge. Clinical signs of infection were significantly reduced in IgG-lavaged infected spinal sites. Bacterial enumeration also exhibited statistically significant reductions in soft tissues, bone and on K-wire spinal implants using IgG lavage compared with saline. Complete healing of all surgical wounds was seen after 28 days, although isolated fibrosed abscesses were observed in autopsied sites treated with both IgG and saline lavages. Local use of IgG wound lavage is proposed as supplementary infection prophylaxis against antibiotic resistant implant-centered or surgical wound infection.

Expression of Cdcrel-1 (Pnutl1), a gene frequently deleted in velo-cardio-facial syndrome/DiGeorge syndrome.

The murine Cdcrel-1 (Pnutl1) gene belongs to the family of septins, which are thought to be involved in cytokinesis in yeast, Drosophila and vertebrates. Recent studies implicate Cdcrel-1 in the regulation of vesicle transport in neurons of the adult brain. The human homologue, hCDCREL-1 maps to chromosome 22q11.2, a region commonly deleted in patients displaying velo-cardio-facial syndrome (VCFS) or DiGeorge syndrome (DGS). During development, Cdcrel-1 transcripts are expressed from E10.5 on in the nervous system such as the dorsal root ganglia and the cranial ganglia as well as the lateral layer of the neural tube, the area where terminally differentiated neurons are located. Low level expression is found in the mesenchyme of the frontonasal mass and the limb bud mesenchyme of E11.5 and E13.5 murine embryos. At E15.5, expression is detected in the nervous tissue and in the neural layer of the eye. Based on the expression pattern as well as clinical data, Cdcrel-1 may be involved in the etiology of VCFS/DGS.

Isolation of lactate dehydrogenase-elevating viruses from wild house mice and their biological and molecular characterization.

Lactate dehydrogenase-elevating virus (LDV) was first identified as a contaminant of transplantable mouse tumors that were passaged in laboratory mice. It has been assumed that these LDVs originated from LDVs endemic in wild house mouse populations. In order to test this hypothesis and to explore the relationships between LDVs from wild house mice among each other and to those isolated from laboratory mice, we have isolated LDVs from wild house mice and determined their biological and molecular properties. We have screened for LDV tissues of 243 wild house mice that had been caught in various regions of North, Central and South America between 1985 and 1994. We were able to isolate LDVs from the tissues of four mice, three had been caught in Baltimore, MD and one in Montana. We demonstrate that the phenotypic properties (ability to establish a long-term viremic infection, low immunogenicity of the neutralization epitope, high resistance to antibody neutralization and lack of neuropathogenicity) of the four wild house mouse LDVs are identical to those of the primary LDVs isolated from transplantable tumors (LDV-P and LDV-vx), which are distinct from those of the neuropathogenic LDV-C. Furthermore, ORF 5 and ORF 2 and their protein products (the primary envelope glycoprotein VP-3P, and the minor envelope glycoprotein, respectively) of the wild house mouse LDVs were found to be closely related to those of LDV-P and LDV-vx. The LDVs caught in Baltimore, MD were especially closely related to each other, whereas the LDV isolated in Montana was more distantly related, indicating that it had evolved independently. The ectodomain of VP-3P of all four wild house mouse LDVs, like those of LDV-P and LDV-vx, possess the same three polylactosaminoglycan chains, two of which are lacking in the VP-3P ectodomain of LDV-C. These results further strengthen the conclusion that the three polylactosaminoglycan chains are the primary determinants of the phenotypic properties of LDV-P/vx.