Viral inactivation in blood and red cell concentrates with benzoporphyrin derivative.

Using both the vesicular stomatitis virus (VSV) and feline leukemia virus (FeLV) as models we have shown that the photosensitizer benzoporphyrin derivative ring A (BPD), when activated with red light (600-700 nm), is effective in eliminating both free virus and virally infected cells from spiked blood products and whole blood drawn from viremic cats experimentally infected with FeLV, under conditions which appear to share red blood cells. The effect of photodynamic therapy on infected lymphocytes, as visualized by scanning electron microscopy, initially appeared as a limited area of tiny holes in the membrane. These holes were subsequently seen to increase in size until the membrane appeared completely decomposed. The red cell membranes however, seem to be undamaged by such photodynamic treatment.     

Inactivation of viruses with photoactive compounds.

The transmission of human immunodeficiency virus (HIV-1) and other enveloped virus by blood transfusion is a major concern. Photosensitive dyes such as hematoporphyrin derivative (HPD), dihematoporphyrin ether (DHE), benzoporphyrin derivatives (BPD), extended ring porphyrins, sapphyrins and texaphyrins, and various cyanines were used with viral cultures to test the feasibility of using those light-excitable dyes to kill virus. A photodynamic flow cell was used to irradiate viral suspensions or viral infected cells in culture media or in whole blood. Herpes virus (HSV-1) was used to screen compounds. Effective compounds were subsequently tested for their ability to kill HIV-1, CMV, and SIV in culture medium and in blood and proved to effectively kill free virus and infected cells at significant viremias. Irradiation was achieved with a filtered xenon light source and/or tunable dye laser. Concentrations of dyes at 10 times viral kill dose were irradiated in blood which was tested for damage to erythrocytes (RBC), platelets, and blood proteins. No damage to RBC, complement factors, and immunoglobulins was evident immediately after photodynamic treatment. Platelet condition is minimally modified with time. Photodynamic treatment of blood appears to be a feasible means of eradicating virus and some protozoans from blood.     

Photophysicochemical consequences of bovine serum albumin binding to non-transition metal phthalocyanine sulfonates.

The interactions between bovine serum albumin (BSA) and sulfonated metallophthalocyanine (MPc) complexes of aluminum (AlPcS(mix)), zinc (ZnPcS(mix)), silicon (SiPcS(mix)), germanium (GePcS(mix)) and tin (SnPcS(mix)) are studied using fluorescence quenching of BSA by MPc complexes. The fluorescence quantum yields of the non-aggregated MPc complexes (AlPcS(mix), GePcS(mix) and SiPcS(mix)) decreased in the presence of BSA, but increased for the aggregated ZnPcS(mix) and SnPcS(mix) complexes. The BSA: MPc conjugates were less stable than the corresponding MPc complexes. The quenching constants were much higher for the non-aggregated complexes. The aggregated nature of the complexes also affected the rate constants (k(F), k(IC), k(ISC)) for the deactivation of the excited singlet state.     

Preservation of resuspended red cell concentrates. Rate of vesiculation and of spontaneous hemolysis

In resuspended red cell concentrates addition of sucrose, mannitol and sorbitol (30 mM final concentration each) to the SAG medium (150 mM NaCl, 50 mM glucose, 1.25 mM adenine) results in a significant reduction of the spontaneous hemolysis of the cells to about 25% after 3 weeks and to about 40% after 6 weeks preservation. Furthermore, in comparison to the SAG medium the vesiculation rate is reduced to about 40% after 3 weeks preservation. Clear cut differences in the effects between the three additives could not be found. The addition of guanosine (1.25 mM final concentration) to the SAG-sucrose or SAG-sorbitol medium has no significant effects on hemolysis and vesiculation.     

The human blood platelet: its derivation from the red blood cell. II. Instantaneous changes in form in the platelet and red cell. A morphologic study

The human blood platelet arises from the interior of the red cell instantaneously as blood is either damaged or disturbed. The platelet starts out as a fine granular structure that rather suddenly changes in form and function with the passage of a very small amount of time.     

An experimental study on photosensitization of human liver cancer cells with sulfonated aluminum phthalocyanine (ALSPC)

The photosensitizing effect of sulfonated aluminum phthalocyanine to human liver cancer cells was studied by determining the kinetics of its cellular uptake, its state of aggregation therein and its photocytotoxicitic effect on these cells. Fluorescence methods were used to measure the cellular uptake in cell extracts and intact cells in monolayer. Results exhibited that the cellular uptake increases with the incubation time of ALSPC and saturates at 24 hrs. This relation was in coincidence with that between ALSPC's photocytotoxic effect and the ALSPC incubation time. Although the average ALSPC concentration in cells is higher than the incubation concentration of ALSPC, laser fluorescence, experiments showed that the fluorescence peak of ALSPC in cells incubated in higher concentration coincides with that of its aqueous solution of low concentration, suggesting that ALSPC in cancer cells exists in monomer state. Characteristics of the photodamaging effect of ALSPC to cancer cells were studied by both the method of typen blue staining and that of isotope labeling, which showed similar results. Results of the isotope labeling experiments implies that the DNA is damaged in ALSPC photosensitization. The photodamage on cells was also studied with 3 T 3 mouse cells (conversion), showing agreeable results to that with liver cancer cells, which suggests that ALSPC's photocytotoxic effect is nonselective to cell types.     

Inactivation of stable viruses in cell culture facilities by peracetic acid fogging

Looking for a robust and simple method to replace formaldehyde fumigation for the disinfection of virus-handling laboratories and facilities, we tested peracetic acid fogging as a method to inactivate stable viruses under practical conditions. Peracetic acid/hydrogen peroxide (5.8%/27.5%, 2.0 mL/m3) was diluted in sufficient water to achieve ≥ 70% relative humidity and was vaporized as <10 μm droplets in a fully equipped 95 m3 laboratory unit. High titers of reovirus 3, MVM parvovirus and an avian polyomavirus were coated on frosted glass carriers and were exposed to the peracetic acid fog in various positions in the laboratory. After vaporization, a 60 min exposure time, and venting of the laboratory, no residual virus was detected on any of the carriers (detection limit <1 infectious unit/sample volume tested). The log reduction values were 9.0 for reovirus, 6.4 for MVM parvovirus, and 7.65 for the polyomavirus. After more than 10 disinfection runs within 12 months, no damage or functional impairment of electrical and electronic equipment was noted.     

Cryopreservation of buffy-coat-derived platelet concentrates in dimethyl sulfoxide and platelet additive solution

Platelets prepared in plasma can be frozen in 6% dimethyl sulfoxide (Me₂SO) and stored for extended periods at −80 °C. The aim of this study was to reduce the plasma present in the cryopreserved product, by substituting plasma with platelet additive solution (PAS; SSP+), whilst maintaining in vitro platelet quality. Buffy coat-derived pooled leukoreduced platelet concentrates were frozen in a mixture of SSP+, plasma and 6% Me₂SO. The platelets were concentrated, to avoid post-thaw washing, and frozen at −80 °C. The cryopreserved platelet units (n = 9) were rapidly thawed at 37 °C, reconstituted in 50% SSP+/plasma and stored at 22 °C. Platelet recovery and quality were examined 1 and 24 h post-thaw and compared to the pre-freeze samples. Upon thawing, platelet recovery ranged from 60% to 80%. However, there were differences between frozen and liquid-stored platelets, including a reduction in aggregation in response to ADP and collagen; increased CD62P expression; decreased viability; increased apoptosis and some loss of mitochondrial membrane integrity. Some recovery of these parameters was detected at 24 h post-thaw, indicating an extended shelf-life may be possible. The data suggests that freezing platelets in 6% Me₂SO and additive solution produces acceptable in vitro platelet quality.     

Interaction of tetrasubstituted cationic aluminum phthalocyanine with artificial and natural membranes

A study of the properties of water-soluble tetrasubstituted cationic aluminum phthalocyanine (AlPcN₄) revealed efficient binding of this photosensitizer to phospholipid membranes as compared with tetrasulfonated aluminum and zinc phthalocyanine complexes. This also manifested itself in enhanced photodynamic activity of AlPcN₄ as measured by the photosensitized damage of gramicidin channels in a planar bilayer lipid membrane. The largest difference in the photodynamic activity of cationic and anionic phthalocyanines was observed in a membrane containing negatively charged lipids, thereby pointing to significant contribution of electrostatic interactions to the binding of photosensitizers to a membrane. Fluoride anions suppressed the photodynamic activity and binding to membrane of both tetraanionic and tetracationic aluminum phthalocyanines, which supports our hypothesis that interaction of charged metallophthalocyanines with phospholipid membranes is mostly determined by coordination of the central metal atom with the phosphate group of lipid.     

Inactivation of animal viruses during sewage sludge treatment.

Using a previously developed filter adsorption technique, the inactivation of a human rotavirus, a coxsackievirus B5, and a bovine parvovirus was monitored during sludge treatment processes. During conventional anaerobic mesophilic digestion at 35 to 36 degrees C, only minor inactivation of all three viruses occurred. The k' values measured were 0.314 log10 unit/day for rotavirus, 0.475 log10 unit/day for coxsackievirus B5, and 0.944 log10 unit/day for parvovirus. However, anaerobic thermophilic digestion at 54 to 56 degrees C led to rapid inactivation of rotavirus (k' greater than 8.5 log10 units/h) and of coxsackievirus B5 (k' greater than 0.93 log10 unit/min). Similarly, aerobic thermophilic fermentation at 60 to 61 degrees C rapidly inactivated rotavirus (k' = 0.75 log10 unit/min) and coxsackievirus B5 (k' greater than 1.67 log10 units/min). Infectivity of parvovirus, however, was only reduced by 0.213 log10 unit/h during anaerobic thermophilic digestion and by 0.353 log10 unit/h during aerobic thermophilic fermentation. Furthermore, pasteurization at 70 degrees C for 30 min inactivated the parvovirus by 0.72 log10 unit/30 min. In all experiments the contribution of temperature to the total inactivation was determined separately and was found to be predominant at process temperatures above 54 degrees C. In conclusion, the most favorable treatment to render sludge hygienically safe from the virological point of view would be a thermal treatment (60 degrees C) to inactivate thermolabile viruses, followed by an anaerobic mesophilic digestion to eliminate thermostable viruses that are more sensitive to chemical and microbial inactivations.     

Inactivation of bacteriophages with cis-platinum(II) diamminedichloride.

The ability of the cis-plantinum(II) diamminedichloride and its hydrolytic products to inactive DNA bacteriophages was examined on the models T2, T4, T4BO1, T3, and lambda. The inactivation of all bacteriophages under study increases gradually during the first 40-90 min of the action of neutral cis-Pt(II) and later passes into an exponential phase. The extent of the region of slower inactviation is larger for osmotically sensitive strains T2 and T4. Inactivation with the hydrolytic products for cis-Pt(II) proceeds exponentially starting from the very beginning and their inactivating effect is higher by 40-80 times than for a comparable concentration of the original complex. The extent of inactivation is not affected with the HCR marker of the host bacteria. The sensitivity to cis-Pt(II) is higher for bacteriophages with a head permeable to salts. An additional inactivation ("after-effect") was observed after dilution of the complex; it can be removed by adding S-aminoisothiuronium dihydrobromide (AET). The results obtained are in good accord with the assumption that inactivation is due to the hydrolytic products arising in the head of bacteriophage.     

Renal excretion of sucrose after transfusion of sucrose containing red cell concentrates

The elimination of sucrose from plasma and the urinary excretion after transfusions of 394 units of sucrose containing red cell concentrates (RCC) to 108 patients has been studied. 70 mmol sucrose corresponding 3 units of RCC were eliminated from the blood plasma to 90% within 3 hours and excreted by kidneys to 55-80% within 12 hours. The rate of excretion depended on the kidney function. The transfusion of RCC resuspended with the sucrose-rich CSD-AG preservation solution (80 mmol sucrose/l RCC) raised the hemoglobin concentration in patient's blood by 0.5-0.6 mmol/l related to 1 unit of RCC.     

S/D灭活血浆内脂包膜病毒及病毒灭活血浆的研究

研究磷酸三丁酯(TNBP)／Triton X-100对血浆内脂包膜病毒的灭活效果。用VSV病毒和Sindbis病毒作指示病毒,加入血浆后再加磷酸三丁酯／Triton X-100,观察病毒的滴度变化及对血浆蛋白的影响。结果发现终浓度各为1％的磷酸三丁酯／Triton X-100在60min内可以灭活血浆内的两种指示病毒,而血浆蛋白的组成和功能变化很小。经层折、超滤后血浆内磷酸三丁酯和Triton X-100的残余量分别低于5μg/ml,表明S／D处理血浆的安全性和治疗作用都很好,其制剂冰冻血浆或冻干血浆可用于临床治疗凝血因子缺乏症,或用作血容量扩张剂。     

The use of inhibitors of platelet activation or protease activity in platelet concentrates stored for transfusion.

During storage of platelet concentrates the platelets show signs of activation, and extracellular protease activity becomes evident in the plasma. The consequences of platelet activation and plasma protease activity are potentially detrimental to the preservation of platelet function in vitro. The earlier use of prostaglandins during preparation of platelet concentrates to increase the harvest of platelets from whole blood did little to improve their shelf-life. Other compounds that sustain elevated cyclic AMP levels or that directly inhibit platelet agonists provide more effective inhibition of platelet activation during storage. Also, the inclusion of general or specific protease inhibitors appears to improve platelet preservation over extended storage periods. These studies demonstrate the possibility of prolonging the shelf-life of platelet concentrates stored at 22 degrees C through the addition of non-toxic formulations of inhibitors of platelet activation and protease activity.