A Feline-Specific Anti-Nerve Growth Factor Antibody Improves Mobility in Cats with Degenerative Joint Disease-Associated Pain: A Pilot Proof of Concept Study.

BACKGROUND: Neutralizing antibodies against nerve growth factor (NGF) are analgesic in rodent models, naturally occurring degenerative joint disease (DJD) pain in dogs, and chronic pain in humans. OBJECTIVES: To evaluate the efficacy of a fully felinized anti-NGF antibody (NV-02) for the treatment of DJD pain and mobility impairment in cats. ANIMALS: Thirty-four client-owned cats with DJD-associated pain and mobility impairment. METHODS: In a placebo-controlled, pilot, masked clinical study, cats were randomized to a single treatment with NV-02 (0.4 mg/kg SC [n = 11] or 0.8 mg/kg SC [n = 12]) or placebo (saline, SC [n = 11]). Activity was measured objectively. Additionally, owners completed clinical metrology instruments (client-specific outcome measures [CSOM] and feline musculoskeletal pain index [FMPI]) on days 0 (screening), 14 (baseline), 35, 56, and 77. A repeated-measures model was used to evaluate the objective activity data. RESULTS: NV-02 significantly increased objectively measured activity overall (P = .017) and at 2 (P = .035), 3 (P = .007), 4 (P = .006), 5 (P = .007), and 6 (P = .017) weeks after treatment. CSOM scores (P = .035) and pain (P = .024) showed a significant effect of treatment 3 weeks after administration. In the treatment group, 83% of the owners correctly identified the treatment administered compared with 45% of owners in the placebo group (P = .013). No treatment-related adverse effects were identified. CONCLUSIONS: These pilot data demonstrate a 6-week duration positive analgesic effect of this fully felinized anti-NGF antibody in cats suffering from DJD-associated pain.

Comparison of the coagulation profile of fatty liver haemorrhagic syndrome-susceptible laying hens and normal laying hens.

1. The rate of thrombin generation in plasma from Fatty Liver Haemorrhagic Syndrome-susceptible laying hens (FLHS, UCD-003) is more rapid than in plasma from age-matched normal Single Comb White Leghorn (SCWL) laying hens. 2. The rate of thrombin generation in plasma was determined by measuring the biological activity of the specific coagulation proteins, Factors V, VII, VIII, IX and X. 3. The higher activity of Factors V, VII and X in FLHS-susceptible laying hens compared with normal SCWL hens remained consistent after plasma lipid concentrations were reduced. 4. Analysis of the fatty acid composition of plasma phospholipids showed that in normal SCWL laying hens phosphatidylethanolamine contained C18:3n3 whereas it contained C20:3n3 in FLHS-susceptible laying hens. 5. The results suggest that alterations in the composition of the phospholipids that are essential cofactors in the biochemical reactions involved in thrombin generation may be a contributing factor in the development of FLHS.

Assessment of factor V, VII and X activities, the key coagulant proteins of the tissue factor pathway in poultry plasma.

1. Assay methods were developed for key components of the tissue factor pathway of blood coagulation, namely Factor V, Factor VII and Factor X. Using these assays, plasma from healthy laying hens, cockerels and broilers was shown to contain functional and equivalent amounts of each of these clotting factors. 2. The plasma activities for Factor V, Factor VII and Factor X can only be accurately determined when chicken tissue factor is used to initiate the coagulation mechanism in poultry plasma. Neither human tissue factor nor rabbit tissue factor forms a fully functional enzyme reactive complex with chicken Factor VII. 3. The overall tissue factor pathway coagulation mechanism was evaluated in plasma from laying hens, cockerels and broilers using the one-stage prothrombin time assay. As long as sufficient tissue factor was used, the overall clotting time results obtained with human recombinant tissue factor were not significantly different from those obtained with chicken tissue factor. 4. We conclude that poultry plasma does possess a fully functional tissue factor coagulation mechanism, but homologous chicken tissue factor must be used for in vitro assays of the components of this pathway.

An evaluation of the process and costs associated with targeted lookbacks for HCV and general notification of transfusion recipients.

BACKGROUND: The Commission of Inquiry on the Blood System in Canada recommended that hospitals notify patients who received blood between 1978 and May 1990 of the risks of contracting HIV (up to the end of 1985 only) and HCV infection. The commission also recommended that patients should be informed of any transfusion received. STUDY DESIGN AND METHOD: General notifications for HIV and HCV for this period were begun in mid-1994. Notification after discharge of transfusions received after May 1990 was begun in 1997. Targeted HCV lookback was performed from 1995 to 1999. RESULTS: Of 21,016 transfusion recipients from January 1978 to May 1990 identified in the general look-back process and believed still alive, 13,549 (64%) were presumed contacted, by registered mail. The overall contact rate for the ongoing notifications (transfusions after May 1990) cannot be accurately determined, as registered mail was not used and a reply not requested. The total cost for these two processes was CAN$373,481, or $13 per patient believed contacted. Most (56%) of this cost was for the conversion to electronic form of paper transfusion records for the period 1978 through early 1984. In the targeted HCV lookback program 1995 through 1999, 94 percent of 256 recipients of specific components identified as likely to have transmitted HCV either were contacted or had died. Of 84 living recipients, 47 (56%) are HCV positive. The last documented potential seroconversion occurred after a transfusion in November 1991, during the period of first-generation EIA testing. If the targeted HCV lookback had been restricted to transfusions after 1987, as the FDA recommended, we would have failed to identify 39 living patients, of whom 21 are HCV positive. The cost per HCV-positive patient notified in the targeted HCV lookback was CAN $4,174. CONCLUSION: The cost of compliance with the com-mission's recommendations was CAN$569,636. Over 28,000 of 36,773 transfusion recipients were notified or presumed notified, and 272 targeted HCV lookbacks to 256 recipients were performed. Performance of this task required the existence of transfusion records back to 1978, conversion of paper records to electronic form, and adequate secretarial and financial support.

Heterogeneous X-ray survival characteristics of lymphocytes in prolymphocytic leukaemia: mathematical analysis distinguishing delayed cell death and true radioresistance.

The survival of non-dividing (G0) leukaemic lymphocytes in culture is generally too short for their radiosensitivity to be quantitatively assessed, since lethally X-irradiated cells may show a long delay before manifestations of cell death ("interphase death") are seen. Counts of surviving cells will therefore include both lethally-hit cells (apparent survivors), and real survivors which have not been lethally hit. Death rates of irradiated leukaemic and normal cells show great variation between individuals, so that comparisons of radiosensitivity between different cell populations based on surviving cell counts at a single time-point are invalid. In this study the supposed radioresistance of prolymphocytic leukaemia lymphocytes was examined in 6 patients with B-cell disease. Survival curves were plotted from serial observations made over several days after graded X-irradiation (0-1000 cGy). We attempted to interpret these radiation responses in terms of their dose dependence (intrinsic radiosensitivity) and time dependence (cell death rate) characteristics using the best-fitting of four mathematical models, all based on classical "single-hit" target theory. The apparent radioresistance shown in 4 cases could be explained by very slow death rates (T1/2 values 55-205 h) of cells proving otherwise radiosensitive (D37 values 38-123 cGy). Genuine radioresistance was found in only 1 case (actual D37 value above 2000 cGy). By ignoring delayed cell death in clinical assessments, pathological lymphocytes could be mistakenly categorised as resistant to elimination by radiotherapy.

Simplified quantitative estimation in vitro of lymphocyte radiosensitivity applied to patients with chronic lymphocytic leukaemia.

Lymphocyte survival changes observed at 1, 2 and 3 days as responses to 3 doses of ionising radiation in vitro (40, 100, and 500 cGy) are analysed by computer according to a simple (single cell population) mathematical model. Intrinsic radiosensitivity, the susceptibility to lethal injury, which is expressed as the D37 value (the radiation dose permitting 37% survival), is estimated separately from the kinetics of subsequent death of lethally-irradiated cells (expressed as their half-life, or t1/2 value). Among the 35 patients with B-cell CLL studied (15 were never treated), both parameters varied widely and independently of one another. t1/2 ranged from 9-200 h and above, D37 from 14-500 cGy or above. Twenty-three patients were deemed 'radiosensitive' (D37 below 110 cGy). D37 level did not correlate with treatment status, mode of treatment, clinical staging (Rai) or lymphocyte count. With some exceptions, D37 remained relatively constant for individual patients with increasing duration of disease or alterations in treatment status. The assay method may prove useful as an aid in predicting response to low-dose splenic irradiation (SI) in CLL.

Transplantation in Manitoba.

1. Renal transplantation can be performed at small regional centers as successfully as at large centers. 2. Immunosuppression should be individualized for the patient thereby avoiding the use of costly and often clinically complicated immunosuppressive regimens. 3. Small centers need to participate in large regional pools in order to give highly presensitized patients a reasonable chance of successful transplantation. 4. Long-term patient compliance is a major problem and requires careful surveillance of patients' adherence to their prescribed therapy. Frequent follow-up also allows for the detection of late rejection episodes which can often be reversed.

The intrinsic radiosensitivity of lymphocytes in chronic lymphocytic leukaemia, quantitatively determined independently of cell death rate factors.

Survival curve shape for lymphocytes X-irradiated in vitro is governed by death rate as well as intrinsic radiosensitivity. We have resolved into these two components the survival curves obtained for CLL lymphocytes by use of a simple mathematical model. A multiple correlation coefficient comparing the predicted with the experimental survival curves was close to unity (0.954-0.999). For 14/18 patients with unequivocal B-cell CLL, the leukaemic (colchicine ultrasensitive) cells behaved as a homogeneous population (D37 0.32-1.28 Gy). This is similar to the more radiosensitive class of lymphocytes of normal blood (believed to include the B cells) and is some 4-fold less than the more radioresistant class (comprising most of the T cells). The lethally hit cells were homogeneous in death rate, which followed first order kinetics. The half-life (range 9-87 h) was, on average, some 50 per cent shorter than the more radiosensitive normal lymphocytes. The remaining four patients constituted a miscellaneous group. From one of these, it can be seen that an excessively slow death rate can give the misleading impression of radioresistance. It is hypothesized that the benefit afforded certain CLL patients treated with low-dose total body irradiation (TBI) or splenic irradiation (SI) may reside, partly, in the sparing of T lymphocytes of the helper type and in accompanying selective elimination (or functional inactivation) of those of the suppressor type.

Colchicine ultrasensitivity of lymphocytes in chronic lymphocytic leukaemia.

It has been previously reported that, in comparison with normal lymphocytes, the lymphocytes in chronic lymphocytic leukaemia (CLL) are ultrasensitive in culture to the cytocidal action of colchicine. In this report the results of 240 colchicine studies in 87 patients with CLL are presented and analysed in terms of the diagnostic, clinical and haematological significance of colchicine ultrasensitivity (CUS) in CLL. All patients with CLL showed very significantly increased CUS of lymphocytes. The CUS did not vary with clinical and haematological status or time and only very rarely with treatment. There was no correlation between % CUS and subsequent course or survival. The results in CLL and in other forms of lymphocytosis and in non-Hodgkin's lymphoma are compared. It is concluded that the test is of considerable diagnostic value, particularly in identifying low-count and treated CLL and in the exclusion of non-CLL lymphocytosis. It appears to have no prognostic value but may have future application in monitoring treatment which selectively eliminates the abnormal cell population.

Inhibition by colchicine of human lymphocytotoxic function: dependence on cell-bound drug level, spontaneous reversibility and antagonism by desacetylcolchicine (DAC).

Colchicine elicits inhibition of spontaneous, PHA-dependent and antibody-dependent forms of lymphocytotoxicity of peripheral blood lymphocytes (PBL) against allogeneic target cells. The findings are that it does so in cell-bound form and to near-maximum effect in the amount of this produced in PBL exposed to it at 10(-6)M concentration for 2 h at 37 degrees C. This represents only a small fraction of the cells' binding capacity, which suggests that it involves sites special in kind (localisation) rather than number (occupied at random). Desacetylcolchicine (DAC) (a known inhibitor of the colchicine-tubulin binding reaction) afforded the PBL protection at concentrations that antagonised the binding of colchicine to them. That DAC itself hardly inhibited PBL function is attributed by inference to a weaker binding affinity making for readier loss of it upon removal of the free drug. It did, however, exhibit a tight form of binding to other, functionally-insensitive cell sites not competed for by colchicine at 100-fold higher concentration. Contrary to the impression lent by other workers' studies (on mouse lymphocytes), colchicine-induced suppression of cytotoxic function is not necessarily irreversible. PBL cultured in drug-free medium gradually lost bound colchicine and they recovered in capacity to express spontaneous and PHA-dependent activity, but not in antibody-dependent activity. The residual cytolytic activity shown by colchicine pre-treated PBL appears in the case of antibody-dependent activity to be truly colchicine resistant; it survived unchanged a 10-fold increase in cell-bound drug level and it cannot be explained as a possible product of recovery. This colchicine-independence may reflect the existence of tubulin/microtubule-independent mechanisms contributing to antibody-dependent activity. Examination of colchicine-treated PBL for membrane fluidity changes, using the probe molecule DPH and the technique of fluorescence polarisation, has yielded negative results, even for cells treated at excessively high colchicine concentration (10(-4)M). All three forms of lymphocytotoxic activity were retained in PBL reconstituted after cryopreservation in liquid nitrogen.

The fatty acyl chain composition of human normal and leukaemic lymphocytes and its modulation by specialised hydrogenation.

Thirty species of fatty acyl chain have been quantitatively identified in human normal peripheral blood lymphocytes (four donors) and lymphocytes circulating in eight patients with chronic lymphocytic leukaemia (CLL). Towards the aim of influencing cell behaviour by lowering membrane fluidity, reaction conditions for catalytic hydrogenation at physiological temperature and pH have been established that effect reduction of the unsaturated species, and preferentially the polyunsaturated forms, but this has not yet been accomplished without killing the cells. That saturation of ethylenic linkages per se is the cause of death is indicated by separate findings showing that the lymphocytes are capable of withstanding hydrogen gas at the requisite high pressure (9 atm.) or exposure alone to the rhodium catalyst [chlorotris (sodium diphenylphosphinobenzene-m-sulphonate)-rhodium(I) tetrahydrate]. It remains feasible that future use of these two agents in combination under milder conditions to produce much lower degrees of hydrogenation than those reported here will permit the cells to survive. Concerning fatty acyl chain composition, the lymphocytes from most of the patients exhibited an inversion in the level of palmitic and stearic acid. A consistently abnormal pattern exhibited by the patients was a rise in oleic acid and a fall in arachidonic acid content. This same alteration has been demonstrated elsewhere in transformed/neoplastic cell types and hence it could well represent phenotypic expression in the CLL lymphocyte of malignant change. Fatty acyl chain composition remained unchanged in lymphocytes reconstituted after cryopreservation in liquid nitrogen.

A mathematical model resolving normal human blood lymphocyte population x-ray survival curves into six components: radiosensitivity, death rate and size of two responding subpopulations.

The analysis was based on observations of survival decrease as a function of dose (range 0-5 Gy (= 500 rad) and time after irradiation in vitro. Since lymphocyte survival is also sensitive to culture conditions the effects of radiation were examined daily up to 3 days only, while survival of control cells remained ca. 90 per cent. The time-dependent changes were resolved as the death rates (first-order governed) of lethally-hit cells (apparent survivors), so rendering these distinguishable from the morphologically identical, true (ultimate) survivors. For 12 blood donors the estimated dose permitting 37 per cent ultimate survival (D37 value) averaged 0.72 +/- 0.18 (SD) Gy for the more radiosensitive lymphocyte fraction and 2.50 +/- 0.67 Gy for the less radiosensitive, each fraction proving homogeneously radiosensitive and the latter identifying substantially in kind with T-type (E-rosetting lymphocytes). The half-life of lethally-hit members of either fraction varied widely among the donors (ranges, 25-104 hours and 11-40 hours, respectively). Survival curves reconstructed by summating the numerical estimates of the six parameters according to the theoretical model closely matched those observed experimentally (range in multiple correlation coefficient, 0.9709-0.9994 for all donors). This signified the absence of any additional, totally radioresistant cell fraction.

Plasma membrane cholesterol regulates human lymphocyte cytotoxic function.

Cell cholesterol is believed to be confined mainly to the plasma membrane. Treatment here of human peripheral blood lymphocytes with cholesterol-free and cholesterol-containing liposomes to effect, respectively, decreases or increases in cholesterol content measureable by chemical analysis, markedly altered effector functions of the cells. Depletion of cholesterol evoked inhibition of spontaneous and phytohemagglutinin-dependent lymphocyte cytotoxicity against allogeneic target cells. Opposite effects resulted from cholesterol enrichment, with PHA-dependent and antibody-dependent cytotoxicities increasing significantly. Treatment, instead, with the known inhibitor of cholesterol biosynthesis, 25-hydroxycholesterol, had suppressive effects like those resulting from lowering the cholesterol level physically by liposome treatment. Our data suggest that the plasma membrane cholesterol content of different categories of lymphocytes in man is both essential and regulatory for their cytotoxic function.

Cell studies in prolymphocytic leukaemia.

A case of prolymphocytic leukaemia, showing several features not yet reported in this disease, is reported. The majority of lymphocytes in the peripheral blood and bone marrow had markers of both B- and T-lymphocytes. The simultaneous presence of receptors for sheep RBC and surface immunoglobulins on individual cells was demonstrated and the endogenous origin of these markers was established. The lymphocytes had some of the functional characteristics seen in chronic lymphocytic leukaemia (CCL). In vitro cell death in the presence of colchicine (colchicine ultrasensitivity) and polystyrene bead column retention were of the same order as seen in CLL. In contrast with the findings in CLL, these cells were markedly radioresistant in vitro. The dominant clinical features--anaemia and constitutional symptoms--appeared to be related to hypersplenism associated with massive splenomegaly. The relevance of these findings is discussed.

A randomized prospective study of cadaver donor pretreatment in renal transplantation.

In a randomized study ww have been unable to demonstrate any beneficial effect of cadaver donor pretreatment with 5 g of methylprednisolone and 7 g of cyclophosphamide on graft function or survival in 22 recipients of pretreated kidneys compared with 30 recipients of nonpretreated kidneys.