White cells protect donor blood against bacterial contamination

The possible beneficial role of white cells (WBCs) in donor blood has been investigated with respect to their capacity to remove bacteria. Preparations of buffy coat and whole blood, containing as well as reduced of WBCs, were inoculated with Staphylococcus epidermidis, S. aureus, Escherichia coli, Pseudomonas aeruginosa, and Propionibacterium species. Upon storage at room temperature, the presence of WBCs resulted in a reduction of the bacterial content. Units inoculated with S. epidermidis and E. coli were completely cleared of bacteria within 5 to 24 hours. On the other hand, S. aureus, after an initial reduction in number, started to multiply. In WBC-reduced units, the initial bacterial content remained unchanged for 5 hours, but the bacteria then exhibited vigorous growth within 48 hours in buffy coat and slower growth in whole blood. Propionibacterium sp. did not grow with or without WBCs. P. aeruginosa did not grow in buffy coat but showed a growth pattern similar to that of S. aureus in whole blood. The presence of WBCs in the donor blood during the first hours after collection thus seems to rid the blood of at least some species of bacteria. These results indicate that it would be favorable not to perform WBC reduction during blood collection and that several hours of contact can be needed to obtain sterility.     

Inducible nitric oxide synthase (iNOS) expression in monocytes during acute Dengue Fever in patients and during in vitro infection

 

Mononuclear phagocytes are considered to be main targets for Dengue Virus (DENV) replication. These cells are activated after infection, producing proinflammatory mediators, including tumour-necrosis factor-α, which has also been detected in vivo. Nitric oxide (NO), usually produced by activated mononuclear phagocytes, has antimicrobial and antiviral activities.

Methods

The expression of DENV antigens and inducible nitric oxide synthase (iNOS) in human blood isolated monocytes were analysed by flow cytometry using cells either from patients with acute Dengue Fever or after DENV-1 in vitro infection. DENV-1 susceptibility to iNOS inhibition and NO production was investigated using N^G-methyl L-Arginine (N^GMLA) as an iNOS inhibitor, which was added to DENV-1 infected human monocytes, and sodium nitroprussiate (SNP), a NO donor, added to infected C6/36 mosquito cell clone. Viral antigens after treatments were detected by flow cytometry analysis.

Results

INOS expression in activated monocytes was observed in 10 out of 21 patients with Dengue Fever and was absent in cells from ten healthy individuals. DENV antigens detected in 25 out of 35 patients, were observed early during in vitro infection (3 days), significantly diminished with time, indicating that virus replicated, however monocytes controlled the infection. On the other hand, the iNOS expression was detected at increasing frequency in in vitro infected monocytes from three to six days, exhibiting an inverse relationship to DENV antigen expression. We demonstrated that the detection of the DENV-1 antigen was enhanced during monocyte treatment with N^GMLA. In the mosquito cell line C6/36, virus detection was significantly reduced in the presence of SNP, when compared to that of untreated cells.

Conclusion

This study is the first to reveal the activation of DENV infected monocytes based on induction of iNOS both in vivo and in vitro, as well as the susceptibility of DENV-1 to a NO production.     

Plasminogen activator inhibitor: a regulator of ancrod-induced fibrin deposition in rabbits

Plasma levels of a fast-acting plasminogen activator inhibitor (PAI), which neutralizes both tissue plasminogen activator (t-PA) and urokinase, are markedly increased in endotoxin-treated rabbits. The ability of this inhibitor to prevent the fibrinolysis that occurs after a thrombogenic stimulus was investigated in a rabbit model. Normal and endotoxin-treated male New Zealand rabbits were infused with ancrod, an enzyme that causes noncrosslinked fibrin formation in vivo. Ancrod stimulated t-PA activity by 90% in normal rabbits and caused hypofibrinogenemia but did not increase PAI levels or induce fibrin deposition in target organs. Rabbits injected with endotoxin (10 micrograms/kg) showed an increase in PAI from less than 1 to 32 U/mL 4 hours later. When ancrod was infused at this time, 90% of the rabbits developed renal fibrin thrombi. Fibrin deposition was recorded in 40% of the rabbits that received a lower dose of endotoxin (1.0 microgram/kg) and had a PAI level of 14 U/ml at the time of ancrod infusion 4 hours later. Fibrin deposition did not occur in the endotoxin-treated rabbits that received normal saline. These data suggest that high levels of PAI inhibit fibrinolysis in vivo, thereby promoting fibrin clot deposition following a thrombogenic stimulus.     

The pharmacokinetics of plasminogen activator inhibitor-1 in the rabbit

The pharmacokinetics of the activated and latent forms of plasminogen activator inhibitor-1 (PAI-1) isolated from HT1080 fibrosarcoma cells (HT1080 PAI-1) and a nonglycosylated form of human PAI-1 isolated from a yeast expression system (rPAI-1) were followed in the rabbit. As assessed by an immunologic assay specific for human PAI-1, guanidine HCI activated HT1080 PAI-1 and rPAI-1 entered the total plasma volume following intravenous bolus administration and exhibited a biphasic clearance pattern. The t1/2s of HT1080 PAI-1 for the initial and beta phases equalled 6.0 and 24.8 minutes, respectively. The t1/2s of rPAI-1 for the initial and beta phases equalled 8.8 and 34.0 minutes, respectively. Similar results were obtained by measuring PAI-1 activity in plasma and with trace amounts of 125I-rPAI-1, suggesting that the above pharmacokinetic behavior could also apply to endogenous PAI-1. The liver was the main site of rPAI-1 clearance. Unactivated, latent PAI-1 exhibited a very different pharmacokinetic profile. Over 80% of latent rPAI-1 cleared from the circulation within 10 minutes (t1/2 = 1.7 minutes). The difference in clearance behavior between activated and latent PAI-1 may be related to the ability of activated PAI-1, but not latent PAI-1, to rapidly form high-molecular-weight complexes with plasma binding factors which were observed in vitro and in vivo. Because PAI-1 could potentially tilt the fibrinolytic balance toward a prothrombotic state, its rapid clearance may represent an important control mechanism governing the circulating levels of this key component of the fibrinolytic pathway.     

Platelet activation and inhibition of malarial cytoadherence by the anti-CD36 IgM monoclonal antibody NL07

The surface glycoprotein CD36 (GPIV) is known to mediate the adhesion of Plasmodium falciparum malaria-infected red blood cells and to be a receptor for extracellular matrix proteins such as collagen and thrombospondin. The murine monoclonal IgM antibody NL07, which is specific for CD36, has now been shown to also be a potent inhibitor of the adhesion of P falciparum malaria-infected red blood cells to C32 melanoma cells. Treatment of platelets with NL07 monoclonal antibody resulted in rapid degranulation, release of ATP and serotonin, increase in [Ca2+]i, and tyrosine phosphorylation of a substrate protein of 130 kD. In about one-half of the experiments, activation with NL07 resulted in the formation of small aggregates of 10 to 30 platelets, whereas in the other half of the experiments, large aggregates were seen similar to those induced by adenosine diphosphate (ADP) and these large aggregates could be converted to the small aggregates by ATP alpha S or by AP-2 or other antibodies against GPIIb and/or IIIa. Microaggregates of 2 to 5 platelets were seen with Glanzmann's platelets that constitutively lack GPIIb/IIIa. Aggregate formation was not seen with heat-treated serum, in the presence of anti C1q antibodies, or when using C5-, C8-, or C9-deficient human sera. Although activation of platelets with purified complement components results in a slow morphologic change without aggregation, involvement of CD36 results in rapid complement-mediated activation leading to formation of small aggregates that is largely independent of GPIIb/IIIa and that, under certain circumstances, proceeds to the formation of large ADP-dependent aggregates.     

Inhibiting glycogen synthase kinase-3 reduces endotoxaemic acute renal failure by down-regulating inflammation and renal cell apoptosis

Background and purpose:

Excessive inflammation and apoptosis are pathological features of endotoxaemic acute renal failure. Activation of glycogen synthase kinase-3 (GSK-3) is involved in inflammation and apoptosis. We investigated the effects of inhibiting GSK-3 on lipopolysaccharide (LPS)-induced acute renal failure, nuclear factor-κB (NF-κB), inflammation and apoptosis.

Experimental approach:

The effects of inhibiting GSK-3 with inhibitors, including lithium chloride (LiCl) and 6-bromo-indirubin-3′-oxime (BIO), on LPS-treated (15 mg·kg⁻¹) C3H/HeN mice (LiCl, 40 mg·kg⁻¹ and BIO, 2 mg·kg⁻¹) and LPS-treated (1 µg·mL⁻¹) renal epithelial cells (LiCl, 20 mM and BIO, 5 µM) were studied. Mouse survival was monitored and renal function was analysed by histological and serological examination. Cytokine and chemokine production, and cell apoptosis were measured by enzyme-linked immunosorbent assay and terminal deoxynucleotidyl transferase-mediated dUTP–biotin nick-end labelling staining, respectively. Activation of NF-κB and GSK-3 was determined by immunostaining and Western blotting, respectively.

Key results:

Mice treated with GSK-3 inhibitors showed decreased mortality, renal tubular dilatation, vacuolization and sloughing, blood urea nitrogen, creatinine and renal cell apoptosis in response to endotoxaemia. Inhibiting GSK-3 reduced LPS-induced tumour necrosis factor-α (TNF-α) and CCL5/RANTES (released upon activation of normal T-cells) in vivo in mice and in vitro in murine kidney cortical collecting duct epithelial M1 cells. Inhibiting GSK-3 did not block TNF-α-induced cytotoxicity in rat kidney proximal tubular epithelial NRK52E or in M1 cells.

Conclusions and implications:

These results suggest that GSK-3 inhibition protects against endotoxaemic acute renal failure mainly by down-regulating pro-inflammatory TNF-α and RANTES.     

Atypical teratoid/rhabdoid tumor of the spine in an adult: case report and review of the literature

Atypical teratoid/rhabdoid tumors (AT/RTs) are rare, malignant brain tumors which occur almost exclusively in infants and young children. There have been only 17 cases of AT/RT in adults reported in the medical literature and the rarity of this tumor makes the diagnosis in adults difficult. We describe a case of an AT/RT of the spinal cord in an adult. A 43-year old woman presented with neck and left upper extremity pain. An MRI demonstrated a mass lesion in the dorsal spinal cord extending from C4 to C6. The patient underwent a C3 through C7 laminectomy. In consultation with senior pathologists at other institutions, the lesion was initially diagnosed as a rhabdoid meningioma. Molecular genetic studies revealed monosomy 22 and loss of expression of the INI1 gene in 22q11.2. Subsequently, immunohistochemical studies revealed the absence of INI1 gene expression in the malignant cells, supporting the diagnosis of AT/RT. The patient underwent three additional surgical procedures for recurrent disease throughout the neuraxis secondary to leptomeningeal spread of the tumor. Despite aggressive surgical resection, adjuvant chemotherapy and radiation therapy, the patient succumbed to the disease two and a half years after her initial presentation. An unrestricted autopsy was performed. To our knowledge, this is the first case of a spinal atypical teratoid/rhabdoid tumor in an adult fully documented with molecular, immunohistochemical, cytogenetic and autopsy findings.     

The prevalence of hepatitis C in survivors of paediatric malignancy

Objective: To assess the prevalence of hepatitis C in 200 patients with paediatric malignancies, surviving in remission more than 5 years from diagnosis, who had received blood product transfusions before 1990 when routine screening of blood products for hepatitis C began.
Method: The second and third generation Abbott Diagnostics ELISA was used to assess hepatitis C seropositivity. Seropositive patients and those with abnormal liver transaminases were assessed by hepatitis C virus RNA polymerase chain reaction (PCR).
Results: A low incidence (4%) of seropositivity for hepatitis C was found in survivors of paediatric malignancy who were transfused prior to routine screening of blood products in this cohort.
Conclusions: All patients identified have evidence of hepatitis and may be at high risk of developing cirrhosis.     

The production of leukaemia inhibitory factor by human endometrium: presence in uterine flushings and production by cells in culture

The concentration of leukaemia inhibitory factor (LIF) was measured in uterine flushings obtained from normal fertile women, from women with unexplained infertility and from women who suffered recurrent miscarriage. In normal fertile women, LIF was not detected in flushings obtained on days luteinizing hormone (LH)+0 to LH+6 of the cycle, but concentrations gradually increased from day LH+7 to a maximum at day LH+12. The amount of LIF in flushings obtained from women with unexplained infertility was significantly lower than in those from normal fertile women on day LH+10 (P < 0.05). The production of LIF by cultured human epithelial and stromal cells was also investigated. LIF was not detectable in the supernatants of cultured stromal cells. Basal LIF production by epithelial cells varied according to the stage in the cycle at which the biopsy was taken. Significantly more LIF was produced by epithelial cells from late proliferative and early secretory endometrium compared with amounts produced by cells from early proliferative (P < 0.001) and late secretory (P < 0.01) endometrium. High doses of progesterone and oestradiol caused a small decrease in epithelial cell LIF production: the combined effect of progesterone and oestradiol (P < 0.01) was greater than the effect of either steroid alone (P < 0.05). The results show, for the first time, the capability of human endometrium to produce LIF in vivo. The fact that maximum LIF concentrations are present at implantation and that decreased concentrations occur in women with unexplained infertility suggest the importance of this cytokine in embryo implantation.