Three-week Intervals Research Articles

Remission of Demyelinating Polyneuropathy With Immunoadsorption, Low Dose Corticosteroids and Anti‐CD20 Monoclonal Antibody

Demyelinating polyneuropathy with anti-myelin associated glycoprotein (anti-MAG) antibodies is an immune mediated disorder characterized by proximal and distal symmetric weakness. Electrophysiological measurements depict features characteristic for demyelination, including prolonged distal latency, retarded conduction velocity, delayed or absent F-waves, and, rarely, partial conduction block. We report on a 65-year-old patient who was diagnosed with demyelinating polyneuropathy and anti-MAG antibodies five years before admission. Despite immunosuppressive agents and extracorporeal therapy (plasmapheresis) the disease progressed as assessed by clinical symptoms and neurological tests. Laboratory results showed an increase of serum immunoglobulin M and anti-MAG antibodies over time. Because of progressive disease we decided to treat the patient with immunoadsorption followed by application of the anti-CD20 antibody, rituximab. Six cycles of selective immunoadsorption were performed over three-weekly intervals with a repetitively used column (Globaffin); each cycle consisted of four consecutive daily treatments. Starting at cycle 4 the anti-CD20 antibody rituximab was administered with 375 mg/m(2) after immunoadsorption. The pretreatment anti-MAG antibody level of 10,000 U/mL, indicating disease activity, initially increased during treatment to a maximum of 30,559 U/mL. However, after completion of the six cycles, the anti-MAG level had decreased to 2348 U/mL; 16 months after the last immunoadsorption cycle the anti-MAG level had increased to 4134 U/mL, while the conduction velocity and compound motor action potentials remained stabile. Immunoadsorption in combination with a monoclonal anti-CD20 antibody in patients with demyelinating polyneuropathy with anti-MAG is effective and can be used an alternative treatment option in patients with progressive disease.

A novel, cremophor-free, polymeric nanoparticle paclitaxel (do/ndr/02) multicenter study in advanced/ metastatic breast cancer

1115 Background: Nanoparticle paclitaxel (NP), DO/NDR/02 is a cremophor-free polymer based formulation of paclitaxel, developed to alleviate the toxicities associated with cremophor component of conventional paclitaxel (CP). Methods: This phase II (N = 194), open label, randomized, three-arm study was conducted in anthracycline failed advanced recurrent/metastatic breast cancer patients. Estrogen/progesterone receptor positive patients were excluded. Sixty-four patients received CP as a 3-hour intravenous (i.v.) infusion at 175 mg/m2 along with standard premedication, 64 patients received a one-hour i.v. infusion of NP at 300 mg/m2 (NP-300) and 66 patients received 220 mg/m2 (NP-220), without premedication. Treatment was administered at three-week intervals for 6 cycles. Efficacy was assessed using Response Evaluation Criteria in Solid Tumours (RECIST) and adverse events were graded as per National Cancer Institute Common Terminology Criteria (NCICTC) version 3. Results: Demographic data revealed that the disease burden with respect to the number of metastatic sites, presence of liver metastasis and raised tumour marker (CA 15.3) levels was higher in the NP-300 arm as compared to NP-220 and CP-175 arms. Other demographic characteristics were comparable in the three arms. Efficacy analysis revealed 40% objective response rate (ORR) each with NP-300 and NP-220, while CP had an ORR of 32.3%. Stable disease was 32.7% and 29.0% for NP-300 and NP-220, respectively; and 27.3% for CP. Progressive disease for NP-300 was 27.3% and NP-220 was 32.7% compared to 38.7% with CP. Hypersensitivity was not observed with NP inspite of absence of premedication. Incidence of neutropenia was 56.3% and 39.4% with NP-300 and NP-220, respectively; and 50% with CP. Grade 4 neutropenia was 21.8%, 12.1% and 14% with NP-300, NP-220 and CP, respectively. There was no grade 4 neuropathy. Grade 3 sensory neuropathy was observed in 12.5% and 1.5% patients with NP-300 and NP-220, respectively and 6.3% with CP. Conclusions: Nanoparticle paclitaxel (DO/NDR/02) can be safely administered as one-hour i.v. infusion without any premedication. Although, this study was not designed as powered study, but the novel formulation has shown improved efficacy and safety. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Dabur Research Foundation Dabur Research Foundation Dabur Research Foundation

Individualized interval shortening in the chemotherapeutic induction treatment of solid tumors (author's transl)

Sequential chemotherapy with vinblastin and bleomycin, as well as adriamycin and cis-platin, was administered to 29 patients with disseminated malignant testicular teratoma. In order to intensify the induction phase, the intervals between drug administrations were individually shortened, with the next course of treatment following directly after the phase of critical leucocyte depression. This method was pursued over four courses of chemotherapy without dose reduction, followed by courses of chemotherapy in a conventional three-weekly rhythm. A response was obtained in 28 patients, 18 of them achieving complete remission. Taking into consideration unfavourable prognostic factors in the treatment group, this result is better than after the same chemotherapy at three-weekly intervals. Induction treatment adapted to leucocyte response carries with it the risk of higher toxicity, but this would seem to be acceptable in view of the improved long-term prognosis. This or similar intensification of the first treatment phase, for a long time used with leukaemias, could also be considered in the management of various chemotherapy-sensitive solid tumours, especially in younger patients.

Are metastatic testicular tumors curable with high-dose chemotherapy and stem-cell rescue?

Although cisplatin-based chemotherapy cures approximately 80% of patientswith newly diagnosed metastatic germ-cell tumours, the outcome in those failing initial chemotherapy is much less favourable and dependent on certainwell-defined clinical factors. Primary salvage options in patients who do not respond to firstline chemotherapy include conventionaldose cisplatin-based regimens, while high-dose chemotherapy (HDCT)with haematopoietic stem cell rescue has been actively investigated in the last two decades, with controversial results. Einhorn and colleagues have retrospectively analysed their experience of tandem HDCT with carboplatin and etoposide in a large series of consecutive men with metastatic testicular cancer that had progressed after receiving cisplatin-containing combination chemotherapy. This study shows 70%and 50% four-year disease-free survival in patients who received HDCT as second-line or third-line or later therapy, respectively.As it is a retrospective review, one may argue that the results are biased by patient selection. This does not seem to be the case, however, as even patients with very poor prognosis achieved longterm disease-free survival – 50% of survivors were classified high-risk by the International Germ Cell Cancer CollaborativeGroup classification and45% had platinum-refractory disease. It is important to note that all patients in this series receivedperipheral-bloodprogenitors as sources of haematopoietic stem cells. This strategy allowed a rapid engraftment, thereby permitting the administration of two courses of highdose carboplatin plus etoposide with planned delays at three-week intervals and acceptable toxicity. In addition, peripheral-blood progenitors were enriched for CD34+ haematopoietic cells, a procedure which may have a role in eliminating possible cancer cells from the graft. The source of stem cells and their ex vivomanipulationmaywell have contributed to the positive results of the study, although there are no evidence-based data to support this hypothesis at present. Results provided by Einhorn et al. are apparently in contradiction with data from two recently published randomised trials that fail to demonstrate a benefit of HDCT over conventional chemotherapy in patients with a poor prognosis, albeit in earlier phases of the disease. Both studies, designed in the early 1990s during an era of great expectations for HDCT, were planned to detect an overoptimistic improvement of event-free survival. The use of bonemarrow stem cells in some patients has resulted in high transplant-relatedmor-

BACOPP-D - An Etoposide-Free Dose-Escalated Polychemotherapy Regimen in Advanced Hodgkin Lymphoma.

Introduction: The development of the escalated BEACOPP regimen let to an improved outcome in patients with advanced Hodgkin Lymphoma (HD9 study of the GHSG). However, the application of high dose etoposide (cumulative 4,8 g/m2 per 8 cycles) seems to be associated with an increased incidence of secondary MDS and AML, respectively. Therefore, the aim of our multicenter pilot study was to evaluate the efficacy and toxicity of the etoposide free as well as dose intensified BACOPP-D protocol.Methods: Since May 2000 a total of 115 patients with Hodgkin Lymphoma (HL) stage IIB, III, and IV were treated with BACOPP-D which included cyclophosphamide 1250 mg/m2 (d1), adriamycin 25 mg/m2 (d1+2), dacarbazine 250 mg/m2 (d1-3), procarbazine 100 mg/m2 (d1-7), prednisolone 40 mg/m2 (d1-14), bleomycin 10 mg/m2 (d8) and vincristine 1,4 mg/m2 (maximum 2 mg, d8) at three-weekly intervals with granulocyte colony-stimulating factor (G-CSF). A consolidating involved field radiation (30 Gy) was performed only in patients who achieved less than CR following chemotherapy. Post-treatment follow-up included PET imaging.Results: Until now 97 patients (median age 35 years, range 17-65; 61 male, 36 female) are assessable for toxicity and treatment outcome. We analyzed the acute toxicity for 728 cycles of BACOPP-D. CTC/WHO grade III/IV haematological toxicities per patient were observed as follows: leukopenia 93%, anemia 39%, and thrombocytopenia 33%. CTC grade III/IV non-haematological side effects included documented infection (4%) and lung toxicity (one patient). A total of 85 patients (88%) achieved complete remission, 9 patients (9%) achieved partial remission, three patients (3%) had progressive disease. At a median observation time of 39 months (0,9-77 months), five patients have relapsed, and nine deaths were documented (4 HL-specific and 3 treatment related deaths, 1 death due to ruptured Meckel diverticulum with peritonitis, one 65 year-old woman died in CR following myocardial infarction). One patient developed a second neoplasia (hypopharyngeal carcinoma in an alcoholic). The overall survival and freedom from treatment failure rates at 39 months were 91% and 85%, respectively. FDG-PET scans after BACOPP-D chemotherapy were performed in 68 of 97 patients. PET scans revealed no increased FDG uptake in 48/68 patients (71%), in 20 patients (29%) increased FDG uptake was detected. In the group of patients with increased FDG uptake, one patient developed progressive disease and four patients relapsed. In the group with PET-negative findings no patient relapsed.Diskussion: BACOPP-D regimen appears as a feasible and effective treatment which induced a complete morphologic and metabolic remission in a high proportion of patients with advanced HL. The treatment was associated with moderate acute toxicity. No secondary AML or MDS occurred until now.

Therapeutic ablation of the infrapatellar fat pad under ultrasound guidance: a pilot study

To describe the technique of ablation of the infrapatellar fat pad using alcohol injection under ultrasound guidance and to assess the efficacy of the procedure in the relief of pain arising from fat pad impingement. Consecutive patients with anterior knee pain and pathology in the infrapatellar fat pad, confirmed on magnetic resonance imaging (MRI), were enrolled in the study. A mixture of alcohol and local anaesthetic was injected under ultrasound guidance, with repeat injections at three-weekly intervals. Twelve patients (seven men, five women), mean age 30.8 years, underwent the procedure. Visual analogue scale pain scores were recorded before treatment and at the end of the treatment course. Before treatment, the mean (+/-SD) pain score was 7.75 (+/-1.14). Patients underwent a mean of four injections (range 2-6). After treatment, the mean pain score was 2.92 (+/-2.61), representing a decrease of 62% (p<0.001). Follow-up data were complete for all 12 patients. Other than short-lived pain at the time of injection, no serious side-effects were encountered. In patients with pain due to inflammation of the infrapatellar fat pad, ultrasound-guided alcohol ablation of the fat pad can provide effective symptom relief. The results of this pilot study indicate that a larger study is warranted to assess the long-term benefits of this well-tolerated procedure.

Spatio-temporal variation of periphyton biomass and accumulation in a temperate spring-fed stream

Spatio-temporal variation of plant populations often can demonstrate synchronous patterns, particularly within highly connected landscapes. Periphyton biomass (chlorophyll a) and net accumulation were measured at five sites in a spring-fed fourth-order stream located in central Pennsylvania with a mixed land-uses watershed (Spring Creek, USA) to characterize longitudinal variation within the stream. Samples were collected at three-week intervals over one year to describe seasonal patterns of periphyton biomass and net production (n = 17 per site). Spring Creek periphyton biomass and net accumulation increased dramatically from the headwaters to downstream (range 10–1,000 mg/m2). The downstream reaches had exceptionally large algal biomass (chlorophyll a > 300 mg/m2) and potential for rapid turnover. Varying degrees of seasonality were observed among the sites, with upstream sites showing more temporal variation but no distinct seasonal pattern. Despite this, large-scale disturbances within the watershed seem to promote synchrony among sites throughout the stream as reflected by close correlations in chlorophyll values (Pearson correlation coefficient r > 0.50).

Effect of an Irinotencan, 5-Fluorouracil, and Leucovorin Combination Chemotherapy (FOLFIRI) in Metastatic Colorectal Cancer

Purpose: Irinotecan is a recently developed active agent in colorectal cancer. The combination of irinotecan and 5-fluorouracil (5-FU)/lecovorin (LV), known as the FOLFIRI regimen, has been approved for patients with metastatic colorectal cancer. The purpose of this study was to assess the efficacy and toxicity of the FOLFIRI regimen in the treatment of metastatic colorectal cancer. Methods: We reviewed the records of 65 patients who had received the FOLFIRI regimen from Jan. 2002 to Dec. 2005. The combination chemotherapy consisted of irinotecan (150~180 mg/m on day 1, 15) as a 2~6 hour infusion followed by bolus infusion of 5-FU (400 mg/m) and continuous infusion of 5-FU (600 mg/m on days 1, 2, 15, 16), concurrently with LV (20 mg/m on day 1, 2, 15, 16) as a 2 hour infusion. Cycles were repeated in three-week intervals. Results: Of the 65 patients who had received the FOLFIRI regimen, 34 were male and 31 cases female. The median age was 54.4 years. The primary tumor sites were the colon in 29 cases (44.6%) and the rectum in 36 cases (56.4%). The metastatic sites were the liver in 33 cases (50.8%), the peritoneum in 21 (32.3%), the lung in 14 (21.5%), a lymph node in 4, and the pelvis in 2. Twenty-seven patients (41.5%) had received the combination chemotherapy as the first line. Of the patients who received more than 3 cycles, complete response was none. Partial responses were 3 (7.1%), stable disease status in 25 cases (59.5%) and progressive disease status in 14 cases (33.3%). The rate of progressive disease status for patients who had received FOLFIRI as the 2nd or the 3rd line were much higher than that of those who had received it as the 1st line chemotherapy. Early stops (<3 cycles) of chemotherapy were due to toxicity, such as nausea, as diarrhea, in 15 of 19 cases (78.9%). Conclusions: The objective response rate of FOLFIRI was 7.1% in metastatic colorectal cancers. Nausea, vomiting, and diarrhea were the main causes of intolerance to the chemotherapy in most of the patients. J Korean Soc Coloproctol 2007; 23:333-337

Estimation of BCL-2 protein in carcinoma of the breast and its clinical correlation in locally advanced breast cancer

The change in expression of apoptotic markers (Bcl-2 and Bax proteins) brought about by various chemotherapeutic regimens is being used for its predictive value for assessing response to neoadjuvant chemotherapy (NACT) in locally advanced breast carcinoma (LABC). (1) Estimation of Bcl 2 expression in LABC, (2) Any change in Bcl 2 expression following chemotherapy in LABC, (3) Any relation of Bcl 2 estimation to changes in size of tumor, nodal status, age, and menopausal status. This was a prospective study of 120 cases of LABC. All cases were subjected to biopsy and the tissue was evaluated immunohistochemically for apoptotic marker Bcl-2 family protein. Three cycles of NACT were given at three-weekly intervals. Modified radical mastectomy was performed and the specimens were re-evaluated for any change in the Bcl-2 family protein. The clinical response and immunohistochemical response were correlated and compared. Coefficient of correlation was calculated by Pearson correlation coefficient (P-value). Clinical response, as measured by reduction in the tumor size, was observed in 81 (67.5%) patients while immunohistochemical response was observed in 67 (55.8%) patients. Correlation between immunohistochemical and clinical response was found to be statistically significant (P = 0.02). Nodal response was seen in 72 (60%) patients. There were no patients in the N o group; 22 (53.7%) of the N 1 patients were down-staged to N o , while 19 (46.3%) remained N 1 . In patients with N 2 disease, 11 (13.9%) were down-staged to N o status, 39 (49.4%) were down-staged to N 1 status, and 29 (36.7%) did not show any response. Immunohistochemical response was observed in 67 (55.8%) patients. Correlation between immunohistochemical and nodal responses was also found to be statistically significant (P = 0.03). This significant positive correlation between clinical and immunohistochemical responses highlights the importance of apoptotic marker Bcl-2 family protein in predicting the response of LABC to NACT.

Diagnosed Skin Diseases Among Migrant Farmworkers in North Carolina: Prevalence and Risk Factors

Skin diseases are common among farmworkers, yet little research documents their prevalence and risk factors. This analysis documents the prevalence of skin diseases among farmworkers in North Carolina, examines variation in the prevalence across the agricultural season, and delineates factors associated with skin disease. Data are from a longitudinal surveillance study with assessments at approximately three-week intervals from May through October 2005. The sample included 304 farmworkers from 45 camps with 1048 data points. Data collection included a structured interview and a standard set often digital photographs. A board-certified dermatologist reviewed the photographs and made specific diagnoses in five categories: inflammatory disease, infection, pigmentary disorder, tumor, and trauma. The prevalences of the five skin disease categories and specific diagnoses are described with counts and frequencies for the entire season and for six time periods. The inflammatory disease and infectious disease categories are modeled with an extension of logistic regression that accounts for repeated measures and clustering of farmworkers within camps. Farmworkers experience high levels of inflammatory skin disease (57.2%) including acne, folliculitis, and contact dermatitis; infectious skin disease (73.8%) including tinea pedis, onychomycosis, and warts; pigmentary disorders (19.1%); and trauma (34.5%). The odds of inflammatory skin disease decreased with age, while those for infectious skin disease increased with age. The odds of inflammatory skin disease increased with pesticide exposure and decreasing housing quality. Skin diseases are highly prevalent among farmworkers. Research is needed to delineate specific factors causing high levels of infection and inflammation in this population.

Expression library immunization confers partial protection against Chlamydia muridarum genital infection

Protective sequences of Chlamydia muridarum were identified as potential vaccine candidates by screening a genomic DNA expression library and assessing the immune responses of mice immunized with individual library clones following vaginal challenge with live Chlamydia. Groups of female BALB/c mice were immunized intra-abdominally by gene gun delivery of DNA three times at three-weekly intervals with individual library clones expressing chlamydial protein fragments and humoral and cell-mediated immune responses were evaluated. Chlamydia-specific cytokines including tumour necrosis factor-α (TNF-α) interleukin-10 (IL-10), interleukin-4 (IL-4), interleukin-12 (IL-12) and interferon-γ (IFN-γ) were detected in mice immunized either with selected DNA clones in spleen cells (0.2–135.2 pg/mL) or lymph nodes (0.15–84.9 pg/mL). The most protective antigen identified was TC0512, a putative outer membrane protein (OMP). Immunization of mice with this clone elicited T-helper type-1 (Th-1) and T-helper type-2 (Th-2) cytokines as well as and IgG1 and IgG2a in sera of these animals. Ten days after the last immunization, animals were challenged intra-vaginally with 5 × 10 4 inclusion-forming units (IFUs) of C. muridarum. At 9 days following challenge TC0512 showed a 73% reduction in the number of recoverable Chlamydia compared with vector only immunized controls. Six additional clones were identified that also conferred varying degrees of protection against live chlamydial challenge. Significant protection against the initial stages of infection was shown by two DNA clones (encoding hypothetical proteins) and five clones showed enhanced clearance of chlamydial infection following DNA immunization and live chlamydial challenge. These results demonstrate that the C. muridarum genome can be screened for individual vaccine candidates by genetic immunization and that the screen produces novel and partially protective vaccine candidates.

Rituximab as an adjuvant therapy to immune tolerance in a haemophilia A boy with high inhibitor titre

We report a haemophilia A boy with high inhibitor titre (170 BU) who experienced five life-threatening bleeding episodes during a one-year period from 9 to 21 months. At the age of 22 months, he received rituximab (375 mg m(-2) per dose) at one- and three-week intervals, three courses each and alternative daily treatment with factor VIII concentrate at doses of 100 units kg(-1) for 24 weeks and 50 units kg(-1) for the following 28 weeks. Although the pretreatment inhibitor level of 4.5 BU showed an anamnestic response reaching the maximum level of 200 BU at the 9th week of treatment, it gradually declined to 30 BU at the 22nd week and was constantly maintained at 25-30 BU for the following 30 weeks. Only three bleeding episodes of two haematomas and one haemarthrosis were found during the one-year treatment period. No opportunistic infection occured during this period.

Preparing clinical-grade myeloid dendritic cells by electroporation-mediated transfection of in vitro amplified tumor-derived mRNA and safety testing in stage IV malignant melanoma

BackgroundDendritic cells (DCs) have been used as vaccines in clinical trials of immunotherapy of cancer and other diseases. Nonetheless, progress towards the use of DCs in the clinic has been slow due in part to the absence of standard methods for DC preparation and exposure to disease-associated antigens. Because different ex vivo exposure methods can affect DC phenotype and function differently, we studied whether electroporation-mediated transfection (electrotransfection) of myeloid DCs with in vitro expanded RNA isolated from tumor tissue might be feasible as a standard physical method in the preparation of clinical-grade DC vaccines.MethodsWe prepared immature DCs (IDCs) from CD14+ cells isolated from leukapheresis products and extracted total RNA from freshly resected melanoma tissue. We reversely transcribed the RNA while attaching a T7 promoter to the products that we subsequently amplified by PCR. We transcribed the amplified cDNA in vitro and introduced the expanded RNA into IDCs by electroporation followed by DC maturation and cryopreservation. Isolated and expanded mRNA was analyzed for the presence of melanoma-associated tumor antigens gp100, tyrosinase or MART1. To test product safety, we injected five million DCs subcutaneously at three-week intervals for up to four injections into six patients suffering from stage IV malignant melanoma.ResultsThree preparations contained all three transcripts, one isolate contained tyrosinase and gp100 and one contained none. Electrotransfection of DCs did not affect viability and phenotype of fresh mature DCs. However, post-thaw viability was lower (69 ± 12 percent) in comparison to non-electroporated cells (82 ± 12 percent; p = 0.001). No patient exhibited grade 3 or 4 toxicity upon DC injections.ConclusionStandardized preparation of viable clinical-grade DCs transfected with tumor-derived and in vitro amplified mRNA is feasible and their administration is safe.

Vaccination of goats with fresh extract from Sarcoptes scabiei confers partial protective immunity

TARIGAN, S. 2006. Vaccination of goats with fresh extract from Sarcoptes scabiei confers partial protective immunity. JITV 11(2): 144-150. Protective immunity has been known to develop in animals infested with Sarcoptes scabiei. However, our previous attempt to induce protective immunity in goats by vaccination with fractions of soluble or insoluble mite proteins had been unsuccessful. Degradation or denaturation of protective antigens occurred during vaccine preparation was suggested as one possible cause of the failure. In this study, mite proteins that used to immunise animals were prepared rapidly in order to prevent protein degradation or denaturation. About 150 mg of freshly isolated mites were rapidly homogenised, centrifuged then separated into supernatant and pellet fractions. Twenty-eight goats were allocated equally into 4 groups. Group-1 goats were vaccinated with the whole mite homogenate supernatant, group 2 with the supernatant, group 3 with the pellet, and group 4 with PBS (unvaccinated control). Vaccination was conducted three times, with three-week intervals between vaccinations, using Quil A as adjuvant, and each vaccination using fresh mite homogenates. One week after the last vaccination, all animals were challenged with approximately 2000 live mites. The severity of lesions, scored from 0 (no lesions) to 5 (>75% infested auricle affected), were determined one day, two days, then every week after challenge. Mite challenge caused the development of skin lesions in all animals. No significant differences between vaccinated and unvaccinated animals were observed in regards to the severity of lesions. However, the mite densities in vaccinated animals were significantly lower (P=0.015) than those in unvaccinated control. This study indicates that the protective antigens of S. scabiei are liable to degradation or denaturation and exist in a very low concentration or have vary low antigenicity. This implies isolation of the protective antigens by the conventional approach, fracionation of the whole mite proteins and testing each fractions in vaccination trials, is seemingly inappropriate for S. scabiei.

The evaluation of extracorporeal shockwave therapy in naturally occurring osteoarthritis of the stifle joint in dogs

Extracorporeal shockwave therapy (ESWT) has expanded from the original uses of human urinary calculi treatment to veterinary orthopaedic applications. This paper investigates the feasibility and efficacy of treating dogs with osteoarthritis of the stifle joint with ESWT. In this study, dogs with persistent stifle lameness despite previous surgical or medical treatment were either treated with ESWT or served as untreated controls. The more lame rear limb of each dog was determined by force platform analysis. The range of motion (ROM) of the stifle joints was assessed by goniometry. Force platform gait analysis and goniometry were performed on both groups for four visits at three-week intervals and a final examination four weeks later. Shock wave therapy was performed three times on the treated dogs, once at each of the first three examinations. A placebo treatment consisting of clipping and wetting the hair was performed on the control dogs. The vertical forces were evaluated for objective analysis of treatment response. For peak vertical force (PVF), four of seven treated dogs improved, while only one of five of control dogs improved. The PVF for the within group analysis did not show any significant change for the treated group, however, the control group has a significant decrease (p = 0.05) in PVF consistent with an increase in lameness. The range of motion (ROM) of the stifle joint improved in five of seven treated dogs and three of five controls. Dogs in the treated group had a trend toward increased ROM (p = 0.07) and a 'positive slope' when compared to dogs in the control group which did not have a significant change (p = 0.78) and had a negative slope indicating the dogs were developing a decrease in ROM. The subjective data provided by client questionnaire did not show significant difference between groups.

Immunotherapy of malignant melanoma using a dendritic cell-amplified tumor RNA vaccine: A phase I study

2597 Background: We prepared a dendritic cell (DC) vaccine specific for malignant melanoma and tested its safety in a phase 1 clinical trial of immunotherapy of stage IV malignant melanoma. Methods: Immature DCs were prepared from immunomagnetically isolated patients’ CD14+ cells. Messenger RNA was isolated from fresh tumor tissue obtained at surgery and was reversely transcribed, amplified, and in vitro transcribed. Amplified RNA was tested for the presence of melanoma-associated antigens gp100, tyrosinase and Mart1 and introduced into immature DCs by electroporation. Then the DCs were matured and cryopreserved. Five million cells were injected at three-week intervals for up to four injections. Results: We studied six patients (median age 52, range 40–66) suffering from predominantly non-visceral disease (four patients). After surgery, the tumor was measurable in three patients, evaluable in two and non-evaluable (completely resected) in one patient. Prior to DC therapy, two patients were treated by radiation, one by hormonal therapy and three by interleukin-2. The mRNA isolate from one patient did not contain any gp100, tyrosinase or Mart1, one isolate contained tyrosinase and four contained all three transcripts, with the presence of these transcripts maintained after amplification. Electroporation and transfection did not affect viability and phenotype of fresh mature DCs, but post-thaw viability was lower in comparison to non-electroporated cells (69 v. 82%; p<0.03). DC injections were tolerated well. We tested the efficiency of vaccination by measuring in vitro reactivity of patients’ T cells before and after DC injections. Both pre- and post-vaccination T cells from one patient secreted IFN-γ upon DC stimulation in vitro while post-vaccination T cells from another patient proliferated upon such stimulation. Conclusions: We demonstrated the feasibility of preparation of viable clinical-grade DCs transfected with tumor-derived mRNA. The vaccine was tolerated well indicating that it can be used in future clinical trials. No significant financial relationships to disclose.

BA Application Frequency and Concentration Effects on Two Indian Hawthorn Cultivars

Abstract Benzyladenine (BA) is a synthetic cytokinin that promotes lateral shoot development in herbaceous and woody ornamentals and may be a viable alternative to mechanical pruning in nursery production. A study was conducted to determine the effects of BA application number, interval and concentration on two Indian hawthorn [Rhaphiolepis Lindl. Cor. Poir. indica (L.) Lindl. (Crataegus indica L.)] cultivars. In spring 2002, actively growing ‘Olivia’ and ‘Eleanor Taber’ received three applications of 2500 or 5000 ppm BA at a one-, two-, or three-week interval. Data collected four weeks after initial treatment (WAT) reflected the effects of one, two, or three BA applications at each concentration. At this time new shoot counts in both cultivars and foliar injury in ‘Olivia’, but not in ‘Eleanor Taber’, increased with increasing number of applications and BA concentration. Data collected 12 WAT reflected the effects of BA application interval and concentration. At this time new shoot counts increased with decreasing application interval in ‘Olivia’, while ‘Eleanor Taber’ formed the most new shoots when BA was applied at a two-week interval. All treated plants sustained minimal to moderate injury to immature foliage. The experiment was repeated in 2003 using 1750 ppm and 3500 ppm BA on ‘Olivia’ Indian hawthorn. Applications were halted at the first sign of foliar injury resulting in plants treated at one, two, and three-week intervals receiving three, two, and two applications, respectively. New shoot counts on ‘Olivia’ treated at the three application intervals were similar, but greater than those on untreated plants. Control plants had a foliar injury rating similar to that of plants treated at a two or three-week interval, but less than that of plants treated at a one-week interval. Growth index was not affected by treatment in either year.

Dose dependency and individual variability in selected clinical, haematological and blood biochemical responses after systemic lipopolysaccharide challenge in cattle

Previous studies have noted that susceptibility to systemic lipopolysaccharide (LPS) exposure seems to differ between individual cows. However, to date inter-individual variation in the response to intravenous injection of LPS has been reported only as an empirical finding, and its existence or extent has never been backed up by statistical analyses. The aim of the present study was therefore to investigate the dose-dependency of clinical, haematological and blood biochemical responses to intravenous LPS injection in dairy cattle and to determine the extent to which these responses differed between individual cows. Eight dairy cows each received three intravenous injections of Escherichia coli LPS (10, 100, and 1000 ng/kg, consecutively) at three-week intervals. All three LPS doses induced clinical, haematological, and blood biochemical responses lasting up to several days. The strength of all of the responses increased significantly with an increasing LPS dose. A statistically significant inter-individual variation was demonstrated for all clinical, haematological, and blood biochemical responses except for serum calcium concentrations. More than half of the statistical variation in white blood cell and thrombocyte counts could be attributed to the individual. The results of this study show that despite the existence of a dose-response relationship between LPS and ensuing clinical, haematological, and blood biochemical responses, the majority of responses to LPS differ significantly in strength and duration from cow to cow.

On-farm welfare assessment in dairy cattle and buffaloes: evaluation of some animal-based parameters

The aim of this study was to evaluate the repeatability of some animal related variables, which could be used in protocols developed for assessing animal welfare at farm level. Recordings were performed in seven dairy farms (four for cattle and three for buffaloes). The animals were observed on three occasions at three-week intervals. The variables collected for each animal were the following: behaviour during milking (stepping and kicking), avoidance distance, lameness and cleanliness. For each farm and each variable repeatability was computed using the Kendall coefficient of concordance (W). In buffalo farms avoidance distance may be considered highly reliable (W > 0.64), whereas in dairy cattle its reliability ranged from medium (W = 0.43 to 0.59) to high (W = 0.64). Behavioural recordings at milking showed that the reliability of stepping was either medium or high for both buffaloes and cattle (W = 0.51 to 0.66 and W = 0.52 to 0.76 for buffaloes and cattle, respectively). Conversely, kicking was less reliable. In cattle farms the reliability for cleanliness ranged from medium (W = 0.51) to high (W = 0.62 to 0.71), whereas, it was not reliable in the sole buffalo farm where this variable was monitored. In cattle farms, the concordance for lameness score was high in two farms (W = 0.62 and 0.66) and moderate in one farm (W = 0.43), whereas no animals displayed lameness in the fourth farm. In all buffalo farms no animals showed lameness. For each species, the Kruskal-Wallis analysis of variance with one factor (farm) was performed to evaluate the effect of farm on recorded variables. For cattle, avoidance distance (P<0.001), stepping (P<0.001), cleanliness (P<0.001) and lameness (P<0.01) were affected by farm, whereas kicking was not significantly different between farms. In buffaloes a significant effect of farm was observed only for avoidance distance (P<0.01). The Mann-Whitney U test performed on data obtained from the farm where both species were present showed that avoidance distance was lower in buffaloes than cattle (P<0.001). This variable is reliable, feasible and able to discriminate among farms. Lameness and cleanliness scores were able to discriminate only cattle farms, whereas these two parameters, albeit feasible, seem to have low significance for buffaloes. Although stepping during milking was reliable and different among cattle farms, its use in on-farm assessment may be difficult because it is more time consuming, thus less feasible.

EFFICACY OF PRAZIQUANTEL AGAINST SCHISTOSOMA HAEMATOBIUM INFECTION IN CHILDREN

A study was performed to determine the efficacy of praziquantel (PZQ) against Schistosoma haematobium. Children (n = 592) infected with S. haematobium received either a single treatment with PZQ (40 mg/kg) or two or three treatments with PZQ at three-week intervals after the initial treatment and efficacy was monitored for nine weeks. Cure rates at three-weeks post-treatment were low (< 50%), suggesting either that worms are killed very slowly or, more likely, that eggs continue to be released from tissues after worm death. Interestingly, a single dose of PZQ showed high efficacy (cure rate > 83% and egg reduction rate > 98%) when assessed from six weeks post-treatment onward. There were no significant differences in cure rates or intensity of infection between the three cohorts at any point in the study, despite the different treatment regimens. Since children were in contact with transmission sites during the study period, the results suggest good efficacy of PZQ against all stages of S. haematobium, including the immature worms.