Activation of hepatic NKT cells and subsequent liver injury following administration of alpha-galactosylceramide

It has been established that alpha-galactosylceramide (alpha-GalCer), a glycolipid, is recognized by natural killer T (NKT) cells together with the monomorphic MHC-like antigen, CD1d, in mice and humans. In this study, we examined how NKT cells are modulated by in vivo administration of alpha-GalCer in mice. When 2 microg (or more)/mouse of alpha(-GalCer was injected i.p., the majority of NKT cells disappeared in the liver and spleen, possibly undergoing apoptosis, on day 1. At this time, NKT cytotoxicity seen in liver lymphocytes also disappeared. In parallel with this numerical and functional change of NKT cells, there was always concomitant hepatocyte damage, as shown by histology and elevated levels of transaminases. Subsequently, the number and function of NKT cells continued to increase from day 3 to day 7. The response seen in hepatic (and splenic) NKT cells did not occur in thymic NKT cells. All these phenomena induced in the liver did not appear in NKT-deficient mice such as beta2-microglobulin(-/-) and CD1d(-/-) mice. These results shed further light on the in vivo interaction between NKT cells and alpha-GalCer in mice.     

Biosynthesis of vitamin B12 by Propionibacterium shermanii IV. Vitamin B12 and porphyrin synthesis in relation to the metabolic activities of the bacterium

The bacterial cell pool contains 16 amino acids. No qualitative change appeared in the amino acid pool at different stages of growth. No amino acids were liberated from the cell pool into the culture medium. Pyruvic and α-ketoglutaric acids were detected in the culture medium and cell pool of Propionibacterium shermanii. Bacterial growth, vitamin B₁₂, and coproporphyrin III synthesis increased with increasing age of the culture. A part of the synthesized porphyrin is released into the culture medium. Glucose, the total nitrogen content of the culture medium, ammonia nitrogen, amino acid nitrogen, methionine and glycine decreased gradually with the increase in the age and the bacterial dry weight of the culture. Succinic acid appeared on the 4^th day and increased with age. Acetic and propionic acids were the only detectable volatile acids. The ratio of propionic to acetic acid was about 3:1 (w/w).     

T lymphocytemediated cytotoxicity in hbsag-positive liver disease

It has been suggested that cellular immune responses to the hepatitis B virus are of importance in the production of liver cell damage in both acute and chronic hepatitis. An assay has now been developed which detects lymphocytes cytotoxic for target cells coated with the hepatitis B surface antigen (HBsAg). The reaction could be blocked by prolonged pre-incubation of lymphocytes with highly purified HBsAg and studies with lymphocyte subpopulations have shown that T lymphocytes were the principle effector cells.

When lymphocytes from twenty-three patients with acute hepatitis were used, cytotoxic T cells were demonstrable during the recovery phase, but not in the first 3 weeks of the illness. However, when these same lymphocytes were extensively washed, cytotoxicity was then detected in all the patients, even at the time of presentation. In patients with HBsAg-positive chronic liver disease the results with the standard assay were largely within the normal range, but again with extensive lymphocyte washing cytotoxicity was detected in all of the patients with untreated chronic active hepatitis and in five out of six with more minor histological lesions. The results in five carriers with normal liver histology were completely different, cytotoxicity remaining undetectable even after the extensive washing procedure.

The results suggest that blocking factors, possibly antigen or antigen–antibody complexes, could be interfering with the detection of sensitized T cells in patients with acute and chronic hepatitis, but that there is a true absence of sensitization to HBsAg in healthy carriers with normal liver histology.

     

Evaluation of PCR for diagnosis of visceral leishmaniasis.

An evaluation of Leishmania PCR was performed with bone marrow, lymph node, and blood samples from 492 patients, 60 positive controls, and 90 negative controls. Results were compared with microscopy results for Giemsa-stained smears. PCR and microscopy of lymph node and bone marrow aspirates from patients with microscopically confirmed visceral leishmaniasis (VL) were equally sensitive. However, in patients clinically suspected of having VL and in whom parasites could not be demonstrated by microscopy, PCR was positive for 12 of 23 (52.2%) lymph node aspirates and 8 of 12 (66.7%) bone marrow aspirates, thus confirming the clinical diagnosis of VL. With PCR on filter paper, Leishmania DNA was detected in the blood of 33 of 47 (70%) patients with confirmed VL and in 2 of 11 (19%) patients suspected of having VL. Positive PCR results were more frequently found for blood samples on filter paper than for samples stored in EDTA. In conclusion, PCR is a more sensitive method than microscopy for the detection of Leishmania in lymph node and bone marrow aspirates, being especially useful for the confirmation of cases of suspected VL. Blood from a finger prick may be used for the initial PCR screening of people suspected of having VL. If the PCR of blood is negative, one should perform PCR with lymph node and/or bone marrow material, because PCR with these materials is more often positive.     

Synthetic organic hard capsule colouring agents: in vitro effect on human true and pseudo-cholinesterases

Hard capsules are made of pure gelatin and small quantities of additives, including colouring agents permitted for use in food. In this study, the effects of three colouring agents (sunset yellow, quinoline yellow and erythrosine) on true and pseudo-cholinesterases (ChE) are assessed in erythrocytes and plasma, respectively. Results indicated that the synthetic compounds affected both true and pseudo ChE activity. The concentration of sunset yellow which caused 50% inhibition (IC50) of true ChE was about 64% that of pseudo-ChE; for erythrosine, IC50 was approximately the same for both true and pseudo-ChE; and for quinoline yellow, IC50 for true ChE was 25% of pseudo-ChE, although its effect on both true and pseudo-ChE was greater than seen with the other two dyes. Inhibitions of both true and pseudo-ChE were of mixed type (competitive and non-competitive). The enzyme-inhibitor dissociation constant (Ki) indicated that quinoline yellow was most potent and erythrosine was least potent out of the three compounds. Inhibition of both true and pseudo-ChE by each of the three dyes was abolished by dialysis, indicating that the effects were reversible.     

A quantitative study on the sacrum of the dog.

The aim of this study was to record sacral bone morphometry that may help in selection of the implant type and proper size in sacroiliac separation. For this reason, sacral lengths and width, the length of each sacral vertebrae, distances between cranial and caudal articular processes, vertical and transversal diameters of the cranial endplate, sacral tuberositas and articular surface areas were obtained from 11 dogs. Additionally, the transverse and vertical diameters of the bony structure and sacral canal were measured from six cross-sections. The data of the study were determined to be representative of the sacral values for average-sized dogs, which was confirmed statistically. The highest value was the sacral width among the linear measurements. The ventral sacral length was longer than the dorsal sacral length. The total lateral area of the sacral wing was measured as 677.46 (142.1)mm2. The transverse diameters of the first sacral vertebra important for screw implantation were 46.02 (4.33)mm and 44.18 (5.29)mm in the first and second cross-sections, respectively.     

A comparative study of the JAM test and 51Cr-release assay to assess the cytotoxicity of dendritic cells on hematopoietic tumor cells

Dendritic cells (DCs) are potent antigen presenting cells and possess a direct anti-tumor cytotoxic ability. Nevertheless, the mechanism of anti-tumor cytotoxicity by DCs and the methods for its evaluation are not fully elucidated. In order to clarify this mechanism of cytotoxicity, we examined the ability of DCs 1) to suppress [³H] thymidine (³H-TdR) uptake by tumor cells; 2) to induce cytolysis on ⁵¹Cr-labeled tumor cells; 3) and to induce DNA fragmentation on ³H-TdR labeled tumor cells (JAM test). Cytolysis and DNA fragmentation are markers of necrotic and apoptotic mechanisms of cytotoxicity in vitro, respectively. DCs inhibited approximately 38.6% to 54.8% of the growth of B4D6, NB4, U937, and Daudi cells as evaluated by the uptake of ³H-TdR. However no cytolysis was verified by ⁵¹Cr-release assay. On the other hand, cytotoxicity rates found using the JAM test ranged from 3 to 81% depending on the cell line and the effector to target cell ratio. The discrepancy of cytotoxicity between ⁵¹Cr-release assay and the JAM test may be due to the phagocytosis of apoptotic tumor cells or the absorption of released ⁵¹Cr by DCs surrounding the target cells. In conclusion, the JAM test was more sensitive than the 4-h and the 10-h ⁵¹Cr-release assay to investigate cytotoxicity mediated by DCs toward hematopoietic tumor cell lines in vitro.     

Incidence and outcome of adenovirus disease in transplant recipients after reduced-intensity conditioning with alemtuzumab.

Adenoviruses are emerging as a major cause of infectious complications after allogeneic transplantation. We evaluated the incidence and outcome of symptomatic adenovirus infection or adenovirus disease after alemtuzumab-based reduced-intensity conditioning in 86 consecutive patients. The overall probability of adenovirus disease was 18.4% (11/86 patients). Five patients died of progressive adenovirus disease, and this was the most important infectious cause of mortality in this cohort. The probability of nonrelapse mortality was 49% in patients with adenovirus disease compared with 25.5% in those without (P=.007). The severity of lymphocytopenia and continuation of immunosuppressive therapy were the most important risk factors for progressive adenovirus disease and death. In contrast, patients who were not receiving immunosuppressive therapy or had had it reduced or withdrawn cleared the virus. We also detected a correlation between the lack of preemptive anti-cytomegalovirus (CMV) therapy for CMV reactivation and the risk of progressive adenovirus disease (P=.05). Our findings highlight the emergence of adenovirus as an important posttransplantation pathogen even after reduced-intensity conditioning and demonstrate the effect of the severity of lymphocytopenia, anti-CMV prophylaxis, and immunosuppressive therapy on the outcome of adenovirus disease.