The train-of-four or double-burst ratios cannot reliably exclude residual neuromuscular block in cats.

This study investigated the relationship between train-of-four (TOF) or double burst (DBS) ratios (T4:T1 or B2:B1) and twitch (T1) or burst (B1) magnitudes during the recovery from rocuronium-induced neuromuscular block in dogs and cats. The main hypothesis was that TOF or DBS ratios recover after the recovery of T1 or B1, and hence high ratio levels are sensitive indicators of restoration of the neuromuscular function. Six anesthetized dogs and six anesthetized cats received 0.5 mg/kg of rocuronium intravenously. The amplitudes of T1 or B1 were measured with mechanomyography during neuromuscular block until the neuromuscular function recovered fully. The TOF or DBS ratio was recorded concurrently. In dogs, recovery of T1 and B1 preceded the recovery of the TOF and DBS ratios, and T1 and B1 were always ≥90% of recovery when the respective ratio reached 0.9. In contrast, T1 was still depressed in 5/6 cats when the TOF ratio reached 0.9. At that moment, T1 was 72.5 ± 19.8% of recovery. Similarly, the DBS ratio returned to 0.9 when B1 was still <90% in 3/6 cats of recovery. The TOF and DBS fade in dogs consistently disappeared after the magnitude of T1 or B1 were restored, and hence, ratios ≥0.9 are a sensitive indicator that the neuromuscular function recovered. Our observation in cats however show that the spontaneous recovery of neither the TOF nor the DBS ratio of 0.9 can reliably exclude residual block, as the magnitude of T1 or B1 was still depressed in several instances.

Bias, limits of agreement, and percent errors between acceleromyography and mechanomyography in anesthetized dogs.

This study compared measurements of neuromuscular function with mechanomyography (MMG) and acceleromyography (AMG) in nine anesthetized dogs receiving 0.1mg/kg vecuronium intravenously. Train-of-four (TOF) stimulation was applied to each ulnar nerve every 15s. The resulting amplitude of the first twitch (T1) and the TOF ratio were measured with both monitors. The baseline TOF ratio (prior to vecuronium), onset time (time of injection to T1<5%), recovery index (time between T1 values of 25% and 75%) and duration of neuromuscular block (injection to TOF 0.9) were recorded. The MMG TOF ratios when the AMG first reached 0.7 (AMG 0.7) and 0.9 (AMG 0.9) during recovery were also recorded. Values were compared with paired tests and individual errors>25% between monitors were identified for each dog. Bias, limits of agreement (LOA) and percentage error (PE) between methods were calculated from Bland-Altman plots for T1 and TOF ratio for the complete data set, and for TOF≥0.7 during recovery. There were no statistical differences in baseline TOF ratio, onset, recovery index, duration, AMG 0.7 and AMG 0.9. Individual errors>25% were evident in onset, recovery index, AMG 0.7 and AMG 0.9. Overall, T1 and TOF ratio had a small bias, wide LOA and PE>100%. Percent error was reduced to 30% when TOF≥0.7 was analyzed. Although there were no statistical differences between MMG and AMG in any variable of interest, individual discrepancies, wide LOA and high PE suggest that these monitors should not be used interchangeably for serial measurements on the same animals.

Identification of equine major histocompatibility complex haplotypes using polymorphic microsatellites.

A system for identifying equine major histocompatibility complex (MHC) haplotypes was developed based on five polymorphic microsatellites located within the MHC region on ECA 20. Molecular signatures for 50 microsatellite haplotypes were recognized from typing 353 horses. Of these, 23 microsatellite haplotypes were associated with 12 established equine leucocyte antigen (ELA) haplotypes in Thoroughbreds and Standardbreds. Five ELA serotypes were associated with multiple microsatellite subhaplotypes, expanding the estimates of diversity in the equine MHC. The strong correlations between serological and microsatellite typing demonstrated a linkage to known MHC class I protein polymorphisms and validated this assay as a useful supplement to ELA serotyping, and in some applications, a feasible alternative method for MHC genotyping in horse families and in population studies.

Characterization of liver T-cell receptor gammadelta T cells obtained from individuals chronically infected with hepatitis C virus (HCV): evidence for these T cells playing a role in the liver pathology associated with HCV infections.

The pathogenic mechanisms involved in viral hepatitis are not completely understood. Evidence suggests that the pathology associated with hepatitis C virus (HCV) and hepatitis B virus (HBV) infections are a result of the immune response in the liver to these viruses. The livers of patients with viral hepatitis have been shown to contain elevated numbers of T cells expressing the gamma/delta form of the T-cell receptor for antigen (TCRgammadelta). In this study, we investigated whether liver biopsy specimens obtained from individuals with viral (HCV and/or HBV) or nonviral hepatitis contained TCRgammadelta(+) T cells that could be expanded in vitro by cytokines. A high percentage of liver biopsy specimens obtained from HCV- and/or HBV-infected individuals contained high numbers of TCRgammadelta(+) T cells. In contrast, T-cell lines generated from liver biopsy tissues obtained from individuals with nonviral hepatitis or from normal controls had no preferential expansion of TCRgammadelta(+) T cells. Liver TCRgammadelta(+) T-cell lines from HCV-infected individuals had high levels of non-major histocompatibility complex (MHC)-restricted cytotoxic activity against different targets including primary hepatocytes and produced interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and interleukin 8 (IL-8) following activation by anti-CD3. Surprisingly, none of these liver TCRgammadelta(+) T-cell lines could recognize any of the structural or nonstructural proteins of HCV and had no cytotoxic activity against cells infected with recombinant vaccinia viruses expressing different HCV proteins. However, the crosslinking of CD81, which has been shown to bind HCV particles and E2, resulted in significant levels of IFN-gamma and TNF-alpha production by liver TCRgammadelta(+) T cells. These results suggest that TCRgammadelta(+) T cells may play a role in the liver pathology of HCV infections.

Crosslinking CD81 results in activation of TCRgammadelta T cells.

CD81 is expressed on most cells and is associated with other glycoproteins, including CD4 and CD8, to form multimolecular membrane complexes. Crosslinking of CD81 on TCRalphabeta(+) T cells results in costimulatory signals that have been proposed to be mediated via CD4 or CD8. In this study, we show that CD81 is also expressed on TCRgammadelta(+)CD4(-)CD8(-) T cells. CD81 crosslinking greatly enhanced anti-CD3 activation of both TCRalphabeta(+) (CD4+ and CD8+) and TCRgammadelta(+) T cells with regard to IFN-gamma production. However, crosslinking of CD81 molecules on TCRgammadelta(+) T cells, in the absence of anti-CD3 stimulation, resulted in cytokine production and enhanced IL-2-induced proliferation, demonstrating that physical association with CD4 or CD8 is not necessary for CD81 signaling. In contrast, crosslinking of CD81 on TCRalphabeta(+) T cells, in the absence of anti-CD3 stimulation, failed to activate these T cells. These results suggest that CD81 signaling may be mediated via a different mechanism(s) in TCRgammadelta(+) versus TCRalphabeta(+) T cells.

Role of NK cells in early host response to chlamydial genital infection.

The cell-mediated immune response has been documented to be the major protective immune mechanism in mice infected genitally with the agent of mouse pneumonitis (MoPn), a biovar of Chlamydia trachomatis. Moreover, there is strong evidence to indicate that gamma interferon (IFN-gamma) is a major effector mechanism of the cell-mediated immune response. Previous studies from this laboratory have also reported that the dominant cell population in the genital tract is the CD4 Th1 population. When experiments were performed by the enzyme-linked immunospot assay, high numbers of cells producing IFN-gamma were found in the genital tract, concomitant with resolution of the infection; however, in addition, an increase in IFN-gamma-producing cells which were CD4(-) was seen early in the infection. Since natural killer (NK) cells produce IFN-gamma and have been found to participate in the early responses in other infections, we hypothesized that NK cells are responsible for early IFN-gamma production in the murine chlamydial model. NK cells were quantified by the standard YAC-1 cytotoxicity assay and were found to appear in the genital tract as early as 12 h after intravaginal infection with MoPn. The cells were confirmed to be NK cells by abrogation of YAC-1 cell cytotoxicity by treatment in vitro and in vivo with anti-asialo-GM1. The early IFN-gamma response could also be depleted by treatment with anti-asialo-GM1, indicating that NK cells were responsible for the production of this cytokine. Of interest was our observation that depletion of NK cells also exacerbated the course of infection in the mice and elicited a Th2 response, as indicated by a marked increase in immunoglobulin G1 antibody. Thus, these data demonstrate that NK cells are not only responsible for the production of IFN-gamma early in the course of chlamydial genital tract infection but are also, via IFN-gamma, a significant factor in the development of the Th1 CD4 response and in the control of the infection.

Protracted Treponema pallidum-induced cutaneous chancres in rabbits infected with human T-cell leukemia virus type I.

In a preliminary study, two of four rabbits infected with human T-cell leukemia virus type I (HTLV-I) demonstrated prolonged primary chancres following superinfection with Treponema pallidum, the causative agent of syphilis. Two rabbits inoculated with 1 x 10(7) HTLV-I-infected human MT-2 cells and two with infected rabbit cells from a line established in this laboratory (RLT-P), developed latent HTLV-I infection as detected by seroconversion 10 weeks after infection and by detection of HTLV-I sequences in the DNA of peripheral blood lymphocytes after amplification by polymerase chair reaction (PCR) 15 weeks after infection. The rabbits remained clinically normal and had normal blood counts. Six months after infection, the four HTLV-infected rabbits and two noninfected controls were challenged by the intradermal inoculation of 1 x 10(6) Treponema pallidum into eight sites on the shaved back. The lesions of two of the HTLV-I-infected rabbits had a time course similar to non-HTLV-I-infected controls and were completely healed by 4 weeks. The lesions of one of the other two rabbits with progressive disease began to heal about 7 weeks after T. pallidum challenge. The cutaneous lesions in the other rabbit remained dark-field positive and became a confluent eschar at 8 weeks; healing only after treatment with penicillin. Four months after the primary challenge none of the six rabbits previously challenged with T. pallidum had developed lesions after rechallenge and thus expressed chancre immunity. These results demonstrate that rabbits with latent HTLV-I infections may have defective cell-mediated immunity.

Identification of a novel human B cell activation antigen involved in B cell growth factor-dependent proliferation.

The B6.4 mAb we present here identifies a novel activation Ag of B cell lineage. The B6.4 mAb was generated by immunizing mice with B cell growth factor (BCGF)-responding BA3 cells, and selected by its capability to block BCGF-induced proliferation of BA3 cells. The inhibitory effect of this antibody on BCGF-dependent proliferation was further confirmed by using normal activated B cells in the presence of exogenous BCGF derived from T cells or B cells. Furthermore, it did not affect IL-2-dependent proliferation of B cells. The expression of the B6.4 Ag, as analyzed by FACS, is restricted to in vitro activated B cells, and remains undetectable on resting B or T cells, PHA-activated T cells, and monocytes. The B6.4 Ag is also expressed on some lymphoblastoid B cell lines and most of the lymphomas and CLL of B cell origin; however, it did not express on pre-B cell ALL and plasma cell leukemias. The B6.4 Ag has a Mr of 35,000 Da, as determined by SDS-PAGE of radiolabeled immunoprecipitates from activated B cells. The B6.4 Ag is detectable on B cells as early as 8 h after activation, and precedes that of the IL-2R or transferrin R. All of these results suggest that the B6.4 Ag is an early activation Ag of B cells and is functionally related to a receptor of BCGF, but not IL-2.

Development and characterization of a human B cell line that responds to B cell growth factor but not interleukin 2.

The human lymphoblastoid cell line we present here proliferated in response to a 14,000 m.w. B cell growth factor (BCGF), and not to interleukin 2 (IL 2). This cell line, designated B-A3, was established by Epstein Barr virus (EBV) transformation of Staphylococcus aureus Cowan I (SAC)-activated spleen B cells, and has been maintained in RPMI 1640 medium complemented with 15% fetal calf serum (FCS) without the addition of other exogenous growth factors. A proliferative response, as measured by [3H]thymidine uptake of B-A3 cells was significantly induced by either commercial IL 2-free human BCGF preparations, or phytohemagglutinin-stimulated mixed lymphocyte culture supernatant at all FCS concentrations used in the assay. The most marked proliferation due to BCGF, however, was observed in the absence of FCS. This BCGF-induced proliferation was not influenced by IL 2 or interferon-gamma (IFN-gamma), because both recombinant IL 2 and IFN-gamma failed to induce proliferation. The response of B-A3 cells to a specific BCGF was additionally indicated by the responsiveness of this cell line to BCGF purified by a series of chromatographic steps. The BCGF to which B-A3 cells responded had a m.w. of 14,000 and was similar to low m.w. BCGF reported from other laboratories. Surface characterization of B-A3 cells, analyzed by flow cytometry with a panel of monoclonal antibodies, demonstrated that the majority of B-A3 cells were stained positively with Leu-12, HLA-DR, and surface IgG markers, whereas staining with surface IgM, IgD markers, pan T cell markers (Leu-4 and Leu-9), and IL 2 receptor (Tac) were consistently negative. Taken together, the human lymphoblastoid cell line we present here responded specifically to a low m.w. BCGF. This cell line may be of value in the purification of BCGF to homogeneity, in studies of the interactions of BCGF with human B cells, and in the identification of the BCGF receptor.

The percentage of T- and B-cells in the lymphomyeloid tissue of hyperimmunized bursaless chickens.

Bursa or bursa follicles failed to develop when 3-day-old embryonated eggs were dipped in 2% testosterone propionate in ethanol. The chicks from the treated and control eggs (TP-chicks) were hyperimmunized with sheep red blood cells and the percentages of thymus-derived (T) and bursal-derived (B) lymphocytes in different lymphoid organs determined. The percentage of T-cells in the thymus of TP-embryos was not significantly affected, but the B-cell population was significantly depressed. The percentage of B-cells in peripheral blood, spleen, and bone marrow of TP-hyperimmunized chickens was significantly below that of controls. These data support previous observations of B-cells in bursaless birds. The significantly higher percentage of T-cells in the bone marrow of TP-birds is discussed in relation to T-suppressor cells.