Forensic veterinary pathology, today's situation and perspectives.

To investigate the current status of forensic veterinary pathology, a survey was composed directed at pathology laboratories and institutes, mostly in Europe. The questions included number of and type of cases, resources available, level of special training of the investigating pathologists and the general view on the current status and future of the discipline. The surveys were sent to 134 laboratories and were returned by 72 respondents of which 93 per cent work on forensic pathology cases. The results indicate scarcity of training opportunities and special education, and insufficient veterinary-specific reference data and information on forensic analyses. More cooperation with human forensic pathology was desired by many respondents, as was more interaction across country borders.

Leishmania donovani requires functional Cdc42 and Rac1 to prevent phagosomal maturation.

Leishmania donovani promastigotes survive inside macrophage phagosomes by inhibiting phagosomal maturation. The main surface glycoconjugate on promastigotes, lipophosphoglycan (LPG), is crucial for survival and mediates the formation of a protective shell of F-actin around the phagosome. Previous studies have demonstrated that this effect involves inhibition of protein kinase C alpha. The present study shows that functional Cdc42 and Rac1 are required for the formation of F-actin around L. donovani phagosomes. Moreover, we present data showing that phagosomes containing LPG-defective L. donovani, which is unable to induce F-actin accumulation, display both elevated levels of periphagosomal F-actin and impaired phagosomal maturation in macrophages with permanently active forms of Cdc42 and Rac1. We conclude that L. donovani engages Cdc42 and Rac1 to build up a protective coat of F-actin around its phagosome to prevent phagosomal maturation.

Role of protein kinase C alpha for uptake of unopsonized prey and phagosomal maturation in macrophages.

Protein kinase C alpha (PKC alpha) participates in F-actin remodeling during phagocytosis and phagosomal maturation in macrophages. Leishmania donovani promastigotes, which inhibit phagosomal maturation, cause accumulation of periphagosomal F-actin instead of the disassembly observed around other prey [Cell. Microbiol. 7 (2001) 439]. This accumulation is induced by promastigote lipophosphoglycan (LPG), which has several effects on macrophages including inhibition of PKC alpha. To investigate a possible connection between PKC alpha and LPG's effects on actin dynamics, we utilized RAW264.7 macrophages overexpressing dominant-negative PCK alpha (DN PKC alpha). We found increased cortical F-actin and decreased phagocytic capacity, as well as defective periphagosomal F-actin breakdown and inhibited phagosomal maturation in the DN PKC alpha-overexpressing cells, effects similar to those seen in controls subjected to LPG-coated prey. The results indicate that PKC alpha is involved in F-actin turnover in macrophages and that PKC alpha-dependent breakdown of periphagosomal F-actin is required for phagosomal maturation, and endorse the hypothesis that intracellular survival of L. donovani involves inhibition of PKC alpha by LPG.

Leishmania donovani lipophosphoglycan causes periphagosomal actin accumulation: correlation with impaired translocation of PKCalpha and defective phagosome maturation.

Lipophosphoglycan (LPG) is the major surface glycoconjugate of Leishmania donovani promastigotes. The repeating disaccharide-phosphate units of LPG are crucial for promastigote survival inside macrophages and establishment of infection. LPG has a number of effects on the host cell, including inhibition of PKC activity, inhibition of nitric oxide production and altered expression of cytokines. LPG also inhibits phagosomal maturation, a process requiring depolymerization of periphagosomal F-actin. In the present study, we have characterized the dynamics of F-actin during the phagocytosis of L. donovani promastigotes in J774 macrophages. We observed that F-actin accumulated progressively around phagosomes containing wild-type L. donovani promastigotes during the first hour of phagocytosis. Using LPG-defective mutants and yeast particles coated with purified LPG, we obtained evidence that this effect could be attributed to the repeating units of LPG. LPG also disturbed cortical actin turnover during phagocytosis. The LPG-dependent accumulation of periphagosomal F-actin correlated with an impaired recruitment of the lysosomal marker LAMP1 and PKCalpha to the phagosome. Accumulation of periphagosomal F-actin during phagocytosis of L. donovani promastigotes may contribute to the inhibition of phagosomal maturation by physically preventing vesicular trafficking to and from the phagosome.

Assessment of neutrophil N-formyl peptide receptors by using antibodies and fluorescent peptides.

Enrichment of chemoattractant receptors on the neutrophil surface has been difficult to assess, primarily because of limitations in sensitivity of visualization. Using an ultrasensitive, cooled charge-coupled device camera, we investigated spatial-temporal relationships between N-formyl peptide receptor distribution and directional motility of human neutrophils. Live cells were labeled with fluorescent receptor ligands, i.e., fluoresceinated tert-butyl-oxycarbonyl-Phe-(D)-Leu-Phe-(D)-Leu-Phe-OH (Boc-FLFLF) and formyl-Nle-Leu-Phe-Nle-Tyr-Lys (fnLLFnLYK), while fixed cells were labeled with either fluorescent peptides or monoclonal antibodies. Double labeling of receptors and filamentous actin (F-actin) was done to investigate possible colocalization. N-Formyl peptide receptors on unstimulated cells were randomly distributed. However, on polarized neutrophils, the receptors accumulated toward regions involved in motility and distributed nonuniformly. In fixed neutrophils, antibody-labeled receptors colocalized with the F-actin-rich leading edge whereas peptide-labeled receptors lagged behind this region. We suggest that neutrophils use an asymmetric receptor distribution for directional sensing and sustained migration. A separation between receptors labeled with peptides and those labeled with antibodies reflects two functionally distinct receptor populations at the membrane of motile neutrophils.

Activation of cAMP-dependent protein kinase is necessary for actin rearrangements in human neutrophils during phagocytosis.

We have investigated the role of cAMP and cAMP-dependent protein kinase (cAPK) in neutrophil phagocytosis. Inhibition of cAPK with H-89 reduced complement- and IgG-dependent phagocytosis to 83 and 46%, respectively. Fluorescence intensity measurements of phalloidin-stained actin in neutrophils showed a reduced amount of filamentous actin (F-actin) in pseudopods and around the phagosome in cells treated with H-89 or cAMP-elevating agents (forskolin and rolipram). The amount of phosphotyrosine-containing proteins was also reduced in pseudopods and around the phagosome. Taken together, the data show that cAMP/cAPK regulates F-actin reorganization during receptor-mediated phagocytosis, particularly triggered by IgG-FcR interaction. Our results support the hypothesis that active subcortical reorganization of F-actin is a prerequisite for FcR-mediated phagocytosis, but is less important during CR3-mediated ingestion.

Fusion of Sendai virus and individual host cells and inhibition of fusion by lipophosphoglycan measured with image correlation spectroscopy.

Fusion between Sendai virus (SV) and individual host cells was investigated with confocal laser scanning microscopy (CLSM) and image correlation spectroscopy (ICS). SV was labeled with the fluorescent probe 7-octadecylamino-4-nitrobenz-2-oxa-1,3-diazole (NBD-NH-C18) and was allowed to bind to host cells (HEp-2, BALB-3T3) at 4 degrees C. The effect of lipophosphoglycan (LPG), isolated from Leishmania donovani, on virus fusion was investigated by incorporation of LPG (0, 5, 10 or 20 microM) into the host cell membrane (HEp-2) before addition of SV. LPG did not affect the number of SV bound per cell. After incubation at 37 degrees C for 15 min without LPG, CLSM revealed a redistribution of NBD-NH-C18 from the SV envelope to the host cell membrane and an increase in average fluorescence intensity, indicating dequenching. ICS analysis of images obtained after incubation at 37 degrees C showed an increased mean cluster density to 260% of the value at 4 degrees C, reflecting the disappearance of labeled SV from the cell surface and diffusion of NBD-NH-C18 into the host cell membrane. Preincubation of the cells with LPG inhibited the temperature-induced redistribution and dequenching of NBD-NH-C18 in a concentration-dependent manner, with a total inhibition of fusion at 20 microM LPG. Together, the results demonstrate that CLSM combined with ICS is a powerful tool for studies of fusion of enveloped viruses with individual host cells and that LPG inhibits the fusion process at or before the hemifusion (lipid mixing) stage of SV interaction with cells.

Organization and mobility of CD11b/CD18 and targeting of superoxide on the surface of degranulated human neutrophils.

A monoclonal IgM, specifically recognizing both CD11b and CD18 of human neutrophils, was used to examine the organization and mobility of CD11b/CD18 in the plasma membrane of human neutrophils degranulated by dihydrocytochalasin B (dhCB) treatment and fMet-Leu-Phe (fMLF) stimulation. Subcellular fractionation analysis of untreated or dhCB-treated control neutrophils indicated that 20% of CD11b/CD18 cosedimented with plasma membrane and the remainder with specific granules. In contrast, fMLF stimulation of dhCB-treated cells caused a major reorganization of CD11b/CD18, in which 60-70% of CD11b/CD18 sedimented in dense plasma membrane fractions that were also enriched in superoxide-generating NADPH oxidase activity. Similarly pretreated neutrophils were fixed, immunogold labeled, and examined by scanning electron microscopy. Immunogold particles were distributed uniformly over the symmetrically ruffled surface of unstimulated neutrophils. On dhCB-treated cells, immunogold was mostly uniformly distributed on a smooth membrane with a small percentage of particles lining up into linear arrays. After fMLF + dhCB stimulation, CD11b/CD18 gold label was more abundant on the cell surface and formed large aggregates on polarized membrane protrusions. However, when cells were adhered to an albumin-coated quartz surface and stimulated with fMLF in the presence of dhCB, immunogold was excluded on the articulated and rounded cell body but concentrated on the periphery of adherent lamellae. Fluorescence photobleaching recovery indicated that in unstimulated cells 38 +/- 3% of CD11b/CD18 was mobile (R) with a diffusion constant D of 3.1 +/- 0.3 x 10(-10) cm2/s. Treatment with dhCB raised R and D 24 and 74%, respectively. Stimulation using 1 microM fMLF with dhCB lowered D and R to near control levels. Since NADPH oxidase and CD11b/CD18 cosediment in high-density plasma membrane domains after fMLF + dhCB stimulation, we speculate that a stimulus-induced reorganization of CD11b/CD18 and NADPH oxidase to common membrane domains may occur in fMLF + dhCB-degranulated neutrophils.

The N-formyl methionyl peptide, formyl-methionyl-leucyl phenylalanine (fMLF) increases the lateral diffusion of complement receptor 1 (CR1/CD35) in human neutrophils; a causative role for oxidative metabolites?

The effects of the N-formyl methionyl peptide, formyl-methionyl-leucyl phenylalanine (fMLF) on the lateral mobility of the complement receptor type 1 (CR1/CD35) in glass-adherent human neutrophils were investigated, using fluorescence recovery after photobleaching (FRAP) and confocal microscopy (CSLM). It was found that addition of 0.1-1 microM fMLF increased the diffusion constant (D) of CR1/CD35 to 167-228% of controls. No effect was observed on the receptor distribution or the mobile fraction of receptors. The effect of fMLF on the lateral diffusion of CR1/CD35 could be totally inhibited by addition of pertussis toxon (PD, 250 ng/ml) or of the free radical scavenger enzymes superoxide dismutase (SOD, 2000 U/ml) and catalase (CAT, 200 U/ml), added together the results show that oxidative metabolites produced by neutrophils in response to fMLF can modulate CR1/CD35 diffusion, and indicate a regulatory role for oxygen radicals in phagocytosis.

Assessment of the influence of thyroglobulin (Tg) autoantibodies and other interfering factors on the use of serum Tg as tumor marker in differentiated thyroid carcinoma.

The purpose of the study was to examine the value of a commercial immunoradiometric (IRMA) method for measuring serum thyroglobulin as a tumor marker after treatment for differentiated thyroid carcinoma. A prospective analysis of consecutive serum samples from 53 patients was performed using the IRMA method and a traditional double antibody radioimmunoassay (RIA). The results were compared with those of 100 healthy control subjects and furthermore the method was validated by investigating sera from 24 patients with Hashimoto's thyroiditis positive for thyroglobulin autoantibodies. Finally, in vitro studies of the influence of thyroglobulin autoantibodies on the method were done. The IRMA method had an acceptable analytical precision and was more sensitive than the RIA. It was furthermore less sensitive to the presence of thyroglobulin autoantibodies but it was affected by them, and it showed less unspecific serum effect. Both methods had limitations as tumor marker when the patients had a thyroid remnant, when serum thyrotropin was not suppressed, and in cases of local recurrence. The highest predictive value was found in patients with distant metastases. Thus, in cases of only slightly elevated serum thyroglobulin, the strongest indication for recurrence is still an increasing serum thyroglobulin level within the same patient rather than a single value.

Irradiation of bone metastases in breast cancer patients: a randomized study with 1 year follow-up.

The results from a prospective randomized trial comparing two different radiation schedules for treatment of painful bone metastases in women with recurrent breast cancer are presented. A total of 217 patients with painful bone metastases were randomized to either 30 Grey (Gy) in ten fractions, five fractions a week (5F/W) or 15 Gy in three fractions 2F/W. The effect of treatment was evaluated by pain assessment, the radiological response and the degree of side-effects. The patients were rated at start of treatment and after 1, 3, 6 and 12 months. No difference between the two radiation regimes was found, neither in achieved pain relief, improvement in level of activity and medication, nor was there any difference in radiological response and side-effects from treatment. Both regimes resulted in a significant improvement in both pain score and level of activity 1 month after treatment, an improvement which persisted during the follow-up period. We conclude that 15 Gy given in three fractions 2F/W is as effective as 30 Gy in ten fractions 5F/W, but more convenient to the patient and of less cost to society.

Randomized clinical trial of pressure-controlled inverse ratio ventilation and extracorporeal CO2 removal for adult respiratory distress syndrome.

The impact of a new therapy that includes pressure-controlled inverse ratio ventilation followed by extracorporeal CO2 removal on the survival of patients with severe ARDS was evaluated in a randomized controlled clinical trial. Computerized protocols generated around-the-clock instructions for management of arterial oxygenation to assure equivalent intensity of care for patients randomized to the new therapy limb and those randomized to the control, mechanical ventilation limb. We randomized 40 patients with severe ARDS who met the ECMO entry criteria. The main outcome measure was survival at 30 days after randomization. Survival was not significantly different in the 19 mechanical ventilation (42%) and 21 new therapy (extracorporeal) (33%) patients (p = 0.8). All deaths occurred within 30 days of randomization. Overall patient survival was 38% (15 of 40) and was about four times that expected from historical data (p = 0.0002). Extracorporeal treatment group survival was not significantly different from other published survival rates after extracorporeal CO2 removal. Mechanical ventilation patient group survival was significantly higher than the 12% derived from published data (p = 0.0001). Protocols controlled care 86% of the time. Average PaO2 was 59 mm Hg in both treatment groups. Intensity of care required to maintain arterial oxygenation was similar in both groups (2.6 and 2.6 PEEP changes/day; 4.3 and 5.0 FIO2 changes/day). We conclude that there was no significant difference in survival between the mechanical ventilation and the extracorporeal CO2 removal groups. We do not recommend extracorporeal support as a therapy for ARDS. Extracorporeal support for ARDS should be restricted to controlled clinical trials.

Ethical implications of standardization of ICU care with computerized protocols.

Ethical issues related to the use of computerized protocols to control mechanical ventilation of patients with Acute Respiratory Distress Syndrome (ARDS) are identical to the ethical issues surrounding the use of any therapy or intervention. Four ethical principles must be considered: nonmaleficence, beneficence, autonomy, and distributed justice. The major ethical challenges to computerized protocol use as a specific application of clinical decision support tools are found within the principles of nonmaleficence and of beneficence. The absence of credible outcome data on which ARDS patient survival probabilities with different therapeutic options could be based is a constraint common to most ICU clinical decision making. Clinicians are thus deprived of the knowledge necessary to define benefit and are limited to beneficent intention in clinical decisions. Computerized protocol controlled decision making for the clinical management of mechanical ventilation for ARDS patients is ethically defensible. It is as well supported as most ICU therapy options.

Open lung biopsy does not correlate with pulmonary function after the adult respiratory distress syndrome.

Abnormalities of pulmonary function occur following the adult respiratory distress syndrome (ARDS). To determine if open lung biopsy (OLB) during ARDS predicts late pulmonary function abnormalities, we examined nine survivors of ARDS who had OLB during ARDS. Open lung biopsy was performed within two weeks of the diagnosis of ARDS, and the following were scored by a pulmonary pathologist as to extent and severity: hyaline membranes (HM), interstitial fibrosis (IF), air space organization (AO), interstitial cellularity (IC), and type 2 cell proliferation (T2C). Pulmonary function tests performed at least one year after ARDS were also used for analysis. Percent predicted Dco, TLC, DL/VA, and FVC were regressed against extent, severity, and combined scores. No significant correlation was found despite impressive histologic abnormalities. These data suggest that the severity and extent of HM, IF, AO, T2C, or IC do not correlate with lung function following ARDS.

Nimorazole as a hypoxic radiosensitizer in the treatment of supraglottic larynx and pharynx carcinoma. First report from the Danish Head and Neck Cancer Study (DAHANCA) protocol 5-85.

Between January 1986 and September 1990, 442 patients with pharynx and supraglottic larynx carcinoma were randomized to receive the hypoxic cell radiosensitizer nimorazole (NIM) or placebo in association with a course of conventional primary radiotherapy. A preliminary analysis including the first 288 patients showed that the stratification parameters were significant (3-year actuarial local-regional tumor control, p less than 0.05) for sex (females 52% vs males 34%), tumor size (T1-T2 47% vs T3-T4 32%) and pre-irradiation hemoglobin (Hb) concentration (high 41% vs low 34%). Overall, the NIM group showed a significantly better local-regional control rate than the placebo group (46% vs 32%). There was an apparent additive effect of Hb concentration and NIM. Thus, in the male group, placebo patients with low Hb had a 23% control rate compared to 46% in NIM treated patients with Hb above 9 mmol/l (p less than 0.05). The similar effect in females could not be evaluated due to the small number of women with this disease. NIM was well tolerated and drug-related side effects were minor and tolerable, with transient nausea and vomiting as the most frequent complication. A final conclusion of the study must await an evaluation including all patients and a longer observation time.

[Metastatic spinal compression syndrome. Symptoms, diagnosis, treatment and prognosis]. / Metastatisk spinalt tvaersnitssyndrom. Symptomer, diagnose, behandling og prognose.

A retrospective study of 398 patients suffering from metastatic compression of the spinal cord or cauda equina is presented. The study comprised almost all relevant medical records of patients admitted to hospital in the eastern part of Denmark in the period 1979 through 1985. Carcinoma of the lung, prostate, breast and kidney were the most frequent primary malignancies causing spinal compression. Most patients were treated with laminectomy, or radiotherapy or with laminectomy and radiotherapy combined. The effect of the treatment was estimated by evaluation of motor function and sphincter control. Treatment with laminectomy followed by radiotherapy was significantly superior to treatment with laminectomy or radiotherapy alone. But if the patients' motor function and primary tumour were taken into account, no significant difference between the treatments was observed. The efficacy of treatment depended upon the symptoms when the diagnosis was established, and accordingly early diagnosis is of the utmost importance. The incidence of metastatic compression increased during the period covered by the study, and since this condition must not go untreated, awareness of the symptoms, primarily pain, is essential.

Recovery of gait after radiotherapy in paralytic patients with metastatic epidural spinal cord compression.

We report on 15 patients with paralysis of the legs caused by metastatic epidural spinal cord compression. After radiation therapy 6 patients regained motor function, 5 of whom recovered ambulatory function, but the recovery was delayed for 3 months or more. The duration of the paralysis prior to treatment varied from 20 hours to 10 days with no significant difference between the group with and the group without recovery. The median time from the initial motor symptoms to total paralysis was longer (45 days) in the patients who recovered compared with those permanently paralyzed (9 days). The response to radiation therapy seems to depend on the rate of loss of spinal cord function rather than the length of total paralysis. We recommend active treatment of patients with paraplegia due to metastatic epidural spinal cord compression, even when the paralysis has been present for over a week.

Metastatic epidural spinal cord compression. Results of treatment and survival.

All medical records of patients treated for metastatic compression of the spinal cord or cauda equina in the eastern part of Denmark from 1979 through 1985 were reviewed. With regard to treatment response and survival, 345 patients could be evaluated. Carcinoma of the lung (19%), prostate (18%), breast (13%), and kidney (10%) were the most frequent primary malignancies causing spinal compression. The outcome of treatment depended primarily on the patients' condition at the time of diagnosis: 79% of the patients who were able to walk before the treatment remained ambulatory, whereas only 21% of the nonambulatory paraplegic patients and 6% of the paralytic patients regained walking ability. Patients treated with laminectomy followed by radiotherapy seemed to respond better than patients treated with radiotherapy or laminectomy alone, but when the patients' pretreatment motor function was taken into account no significant difference was found between the three forms of treatment. In the subgroup of nonambulatory patients, however, a significantly better restoration of gait was observed in patients treated with the combination of laminectomy and radiotherapy than in patients treated with radiotherapy alone. A longer survival in the group treated with the combination of laminectomy and radiotherapy may reflect that these patients were in a lower stage of disease and thus had a better potential of regaining motor function. The results call for prospective randomized studies.

Metastatic spinal cord compression. Occurrence, symptoms, clinical presentations and prognosis in 398 patients with spinal cord compression.

We reviewed all medical records concerning patients suffering from spinal cord or cauda equina compression (SCC) secondary to cancer, in the eastern part of Denmark, from 1979 through 1985. During the period the incidence of SCC in cancer patients went up from 4.4% to 6%. However, this increase was not significant. The series comprised 398 cases, with carcinoma of the prostate (19%), lung (18%), breast (14%) and kidney (10%) accounting for 61%. The symptoms were evaluated in accordance with the patients rating of pain, motor deficits, sphincter control and paraesthesia, whereas the clinical manifestations were classified on the basis of motor deficit and bladder dysfunction. During the period preceding the diagnosis of SCC, 83% of the patients suffered from back pain, 67% from deteriorating gait and 48% had retention of the urine. In 35% of the patients there was no sphincter disturbance and 10% had normal sensory function. The outcome of treatment was estimated by changes in motor deficits and sphincter function, and depended primarily on the patients condition at the time of the diagnosis. Of the patients who were able to walk before treatment, 79% remained ambulatory, whereas only 18% of the non-ambulatory patients regained walking ability. Patients treated by decompressive laminectomy followed by radiotherapy apparently had a better response than patients treated with surgery or irradiation alone, but when the patients pre-treatment motor function was taken into account, no significant difference was observed. The study may call for a properly randomized trial with careful stratification of tumour biology, performance status and neurological deficits.