Inactivation of human immunodeficiency virus type 1 by porphyrins

We have evaluated the anti-human immunodeficiency virus (HIV) activity of a series of natural and synthetic porphyrins to identify compounds that could potentially be used as microbicides to provide a defense against infection by sexually transmitted virus. For assays we used an epithelial HeLa-CD4 cell line with an integrated long terminal repeat-beta-galactosidase gene. For structure-activity analysis, we divided the porphyrins tested into three classes: (i) natural porphyrins, (ii) metallo-tetraphenylporphyrin tetrasulfonate (metallo-TPPS4) derivatives, and (iii) sulfonated tetra-arylporphyrin derivatives. None of the natural porphyrins studied reduced infection by more than 80% at a concentration of 5 micro g/ml in these assays. Some metal chelates of TPPS4 were more active, and a number of sulfonated tetra-aryl derivatives showed significantly higher activity. Some of the most active compounds were the sulfonated tetranaphthyl porphyrin (TNapPS), sulfonated tetra-anthracenyl porphyrin (TAnthPS), and sulfonated 2,6-difluoro-meso-tetraphenylporphine [TPP(2,6-F2)S] and its copper chelate [TPP(2,6-F2)S,Cu], which reduced infection by 99, 96, 94, and 96%, respectively. Our observations indicate that at least some of these compounds are virucidal, i.e., that they render the virus noninfectious. The active compounds were found to inhibit binding of the HIV type 1 gp120 to CD4 and also to completely inhibit the ability of Env proteins expressed from recombinant vectors to induce cell fusion with receptor-bearing target cells. These results support the conclusion that modified porphyrins exhibit substantial activity against HIV and that their target is the HIV Env protein.     

Inactivation of free and cell-associated human immunodeficiency virus in platelet suspensions by aminomethyltrimethylpsoralen and ultraviolet light

BACKGROUND: It has previously been reported that 40 micrograms per mL of aminomethyltrimethylpsoralen (AMT) plus 2.4 to 7.2 J per cm2 of ultraviolet A (UVA) light inactivated 4 to 6 log10 of several model viruses in platelet suspensions. This inactivation was achieved while satisfactory levels of platelet count, pH, morphology, aggregation, and hemostatic effectiveness were maintained. STUDY DESIGN AND METHODS: The efficacy of this procedure for inactivating free and intracellular human immunodeficiency virus (HIV), including integrated proviral sequences, was studied. RESULTS: The kinetics of inactivation for free HIV (4-5 log10 kill with 1.2-4.8 J/cm2) were similar to those obtained for the previously studied viruses. For studies on cell-associated virus, H9 cells productively infected with HIV were added to platelet suspensions and treated with the above regimen of AMT and UVA. The phototreated cells were then cocultivated with uninfected H9 cells for 4 weeks and supernatants were assayed by enzyme-linked immunosorbent assay for HIV p24. No evidence of HIV replication was detectable for cells receiving as little as 2.4 J per cm2 of UVA irradiation in the presence of AMT. Further, it has been demonstrated that stably integrated sequences from the HIV proviral env gene can no longer be amplified by polymerase chain reaction after 1.2 J per cm2 of UVA (with 40 micrograms/mL AMT) exposure. CONCLUSION: These data suggest that AMT and UVA is an effective antiviral treatment for free and cell- associated HIV in platelet suspensions.     

Inactivation by phthalocyanine photosensitization of multiple forms of human immunodeficiency virus in red cell concentrates

BACKGROUND: The use of phthalocyanines in conjunction with red light has been shown to inactivate model lipid-enveloped viruses in red cell concentrates. The ability of this treatment to inactivate multiple forms of human immunodeficiency virus (HIV) was evaluated in this study. STUDY DESIGN AND METHODS: The phthalocyanines used were aluminum phthalocyanine tetrasulfonate (AIPcS4) and the silicon phthalocyanines HOSiPcOSi(CH3)2(CH2)3 N(CH3)2 (Pc 4), and HOSiPcOSi(CH3)2 (CH2)3N+(CH3)3I-(Pc 5). HIV was studied in a cell-free form, in an actively replicating form, in latently infected cells, and in blood from HIV-positive patients. RESULTS: All three phthalocyanines inactivate > or = 10(5) infectious doses of cell-free HIV. However, only Pc 4 effectively inactivated actively replicating HIV and latently infected cells. The latter was about four times as sensitive to inactivation as was actively replicating HIV. Increasing the hematocrit of red cells during treatment decreased the rate of inactivation, especially at lower light doses. Under treatment conditions that completely inactivated the laboratory isolates of HIV, cell-associated HIV in blood from HIV-positive patients was also completely inactivated. The polymerase chain reaction signal from the gag gene of HIV was not affected on treatment of cell-free virus, but it was reduced after treatment of cell-associated HIV, particularly in some latently infected cell lines. CONCLUSION: Pc 4 and red light are effective in eliminating the infectivity of HIV in red cell concentrates. The usefulness of this approach for blood banking depends on future demonstration of the preservation of red cell circulatory survival and function in vivo.     

噻唑橙光化学法对血浆中病毒灭活作用的研究

目的探讨噻唑橙(TO)光化学法对血浆中伪狂犬病毒(PRV)和Sindbis病毒的灭活效果。方法配制TO-血浆溶液并测量其吸收光谱,制作照射光源;以PRV和Sindbis病毒为指示病毒,加入血浆以模拟病毒污染血浆;测量病毒灭活动力学曲线;做TO浓度(C)、光照强度(C)、光照时间(T)三因素三水平正交试验(n=4)以确定最优灭活条件;验证最优条件下裸照、血袋充氧前后的病毒灭活效果。结果 TO-血浆最大吸收峰位于476 nm处;病毒灭活动力学曲线显示:在TO 60μmol/L、光强1.06E-01 w.m-2.nm-1条件下,时间5 min,即可将血浆中绝大多数病毒灭活,此后进入平台期;正交试验显示:血浆中PRV及Sindbis病毒最优灭活条件均为:I=1.33E-01 w.m-2.nm-1,T=20 min,C=80μmol/L。在此条件下,灭活效果为裸照PRV≥6.13 LogTCID50(n=8),Sindbis病毒为(5.86±0.29)LogTCID50(n=8);血袋PRV(5.66±0.29)LogTCID50(n=4),Sindbis病毒(3.88±0.25)LogTCID50(n=4);充氧血袋PRV(6.32±0.55)LogTCID50(n=4),Sindbis病毒(6.00±0.35)LogTCID50(n=4)。结论 TO光化学法可有效灭活血浆中病毒,氧含量的增加可以提高病毒灭活的效果。     

Inactivation of kallikrein in human plasma

Human plasma kallikrein is inactivated by plasma protease inhibitors. This study was designed to determine the nature of these protease inhibitors and to assess their relative importance in the inactivation of kallikrein. Therefore, the kinetics of kallikrein inactivation and the formation of kallikrein inhibitor complexes were studied in normal plasma and in plasma depleted of either alpha 2-macroglobulin (alpha 2M), C1 inhibitor, or antithrombin (AT III). Prekallikrein was activated by incubation of plasma with dextran sulfate at 4 degrees C. After maximal activation, kallikrein was inactivated at 37 degrees C. Inhibition of kallikrein amidolytic activity in AT III-deficient plasma closely paralleled the inactivation rate of kallikrein in normal plasma. The inactivation rate of kallikrein in alpha 2M-deficient plasma was slightly decreased compared with normal plasma, but in contrast to normal, C1 inhibitor-deficient, and AT III-deficient plasma, no kallikrein amidolytic activity remained after inactivation that was resistant to inhibition by soybean trypsin inhibitor. Suppression of kallikrein activity in C1 inhibitor-deficient plasma was markedly decreased, and this was even more pronounced in plasma deficient in both C1 inhibitor and alpha 2M. The pseudo first-order rate constants for kallikrein inactivation in normal, AT III-deficient, alpha 2M-deficient, C1 inhibitor-deficient plasma, and plasma deficient in both alpha 2M and C1 inhibitor, were 0.68, 0.60, 0.43, 0.07, and 0.016 min-1, respectively. Sodium dodecyl sulfate gradient polyacrylamide slab gel electrophoresis showed that during inactivation of kallikrein in plasma, high-Mr complexes were formed with Mr at 400,000-1,000,000, 185,000, and 125,000-135,000, which were identified as complexes of 125I-kallikrein with alpha 2M, C1 inhibitor, and AT III, respectively. In addition, the presence of an unidentified kallikrein-inhibitor complex was observed in AT III-deficient plasma. 52% of the 125I-kallikrein was associated with C1-inhibitor, 35% with alpha 2M, and 13% with AT III and another protease inhibitor. A similar distribution of 125I-kallikrein was observed when the 125I-kallikrein inhibitor complexes were removed from plasma by immunoadsorption with insolubilized anti-C1 inhibitor, anti-alpha 2M, or anti-AT III antibodies. These results suggest that only covalent complexes are formed between kallikrein and its inhibitors in plasma. As a function of time, 125I-kallikrein formed complexes with C1 inhibitor at a higher rate than with alpha 2M. No difference was observed between the inactivation rate of kallikrein in high-Mr kininogen-deficient plasma and that in high-Mr kininogen-deficient plasma reconstituted with high-Mr kininogen; this suggests that high-Mr kininogen does not protect kallikrein from inactivation in the plasma milieu. These results have quantitatively demonstrated the major roles of C1 inhibitor and alpha 2M in the inactivation of kallikrein in plasma.     

去白细胞输血器对新鲜冰冻血浆中凝血因子的影响

目的　探讨经白细胞过滤器过滤后的新鲜冰冻血浆 ( fresh frozen plasma,FFP)中凝血因子的生物活性变化。方法　随机抽取 A型、B型、O型、AB型新鲜冰冻血浆各 1 0 0 ml× 5袋 ,3 7℃水浴融化 ,在净化台内留取滤过前后血浆样本各 1 ml,使用德国 BE全自动血凝仪测定活化部分凝血酶时间 ( APTT)、凝血酶原时间 ( PT)、凝血酶时间 ( TT)、纤维蛋白原( Fbg)、凝血因子 ( F ∶C)、凝血因子 ( F ∶C)、凝血因子 ( F C)∶水平。结果　过滤前后的新鲜冰冻血浆 APTT、PT、TT、F ∶ C、F ∶ C、Fbg水平的差异均无显著性 ( P>0 .0 5 )。 F ∶ C过滤前后的差异有显著性 ( P<0 .0 5 ) ,但仍在参考值范围内。结论　过滤前后新鲜冰冻血浆中凝血因子的活性差异变化在参考值范围内 ,适用于临床治疗     

Inactivation of hepatitis A virus in plasma products by vapor heating

BACKGROUND: The transmission of hepatitis A virus (HAV) has been associated with the use of a number of solvent/detergent-treated factor VIII concentrates and possibly a factor IX concentrate. These reports have emphasized the necessity of using virus-inactivation methods for plasma products that are capable of inactivating nonenveloped viruses such as HAV. STUDY DESIGN AND METHODS: A simple, highly accurate titration procedure for HAV, which allows extensive kinetic investigations of virus-inactivation procedures, has been developed. This system has now been used to evaluate the efficacy of vapor heating in inactivating HAV after the addition of the virus to a range of human plasma products. RESULTS: It was demonstrated that HAV was significantly more thermostable than other picornaviruses, which reinforced the fact that such viruses cannot be used as model viruses for HAV-inactivation studies. A one-step vapor-heating procedure was demonstrated to inactivate between 5.9 and > 6.3 log10 of HAV in different products. A two-step vapor-heating procedure had the capacity to inactivate between > 8.7 and > 10.4 log10 of HAV. Both procedures were more effective in inactivating HAV than was the pasteurization procedure used for virus inactivation in human albumin solutions. CONCLUSION: These data demonstrate the efficacy of vapor heating in inactivating high-titer HAV after the spiking of plasma products with virus. This study confirms and explains the results of controlled clinical trials and long-term clinical usage with respect to the lack of HAV transmission by such vapor-heated products.     

Epidemiology and transmission of infection by human immunodeficiency virus

The incidence of HIV infection continues to increase in the United States especially among injection drug users and women. HIV is a retrovirus that infects selected cells in the immune system and under certain conditions replicates and forms new virus capable of infecting other host cells. The infection produces a continuum of conditions ranging from an asymptomatic carrier state through a series of mild or early diseases (ARC) to devastating immune deficiency and organ dysfunction (AIDS). The virus is transmitted via sex, blood, and from mother to fetus. Immune physiology, effects of HIV on the immune system, and tests to demonstrate the presence of the infection are included.     

Treatment of radiation proctitis with argon plasma coagulation

BACKGROUND AND STUDY AIMS: Radiation proctitis is a troublesome complication of pelvic irradiation for malignancy. One common and occasionally serious complication is rectal bleeding. Therapeutic options for this condition are limited. This study was undertaken to evaluate the usefulness of argon plasma coagulation (APC) in the treatment of rectal bleeding due to radiation proctitis. PATIENTS AND METHODS: Fifteen patients referred for treatment of rectal bleeding due to radiation proctitis were offered APC during flexible sigmoidoscopy. Data were collected retrospectively to assess patients' response to treatment. Patients were asked to score overall well-being, bleeding, fecal urgency, incontinence, and stool frequency. Transfusion requirements and nadir hemoglobin before and after treatment were also recorded. Matched data were assessed using the Wilcoxon signed-rank test. RESULTS: Rectal bleeding improved significantly after treatment with APC (median pre-treatment score = 3, median post-treatment score = 1; P<0.001). Transfusion requirements ceased in three patients who had previously been anemic. Hemoglobin levels increased from a mean of 108 g/l to 133 g/l in 13 patients. In addition, other parameters of bowel function, including urgency, incontinence, and stool frequency, improved significantly after treatment. Two patients developed rectal strictures after therapy, but these were asymptomatic and treated with rectal dilation. No other complications were observed. CONCLUSION: APC improved patient well-being and significantly reduced rectal bleeding in patients with radiation proctitis. Transfusion requirements were also reduced. APC is useful and safe in patients in whom radiation proctitis is refractory to other treatments.     

Lentivirus vector mobilization and spread by human immunodeficiency virus

The rapid advancement of lentivirus-based gene transfer systems and their demonstrated utility in a variety of in vitro and in vivo settings has heightened the need for assays to evaluate the safety of these vectors prior to human clinical trials. Two major concerns relating to the use of lentivirus-based vectors in a clinical setting are the presence of contaminating replication-competent retroviruses in vector preparations and the efficiency of vector mobilization and spread by wild-type helper virus (rescue). This article describes an in vitro system to study the rescue of lentivirus-based vectors by wild-type HIV. We show that lentivirus-based vectors can be readily rescued from T cell lines and to a lesser extent from primary human lymphocytes by wildtype HIV, resulting in the spread of mobilized vector particles to previously untransduced cells. Furthermore, we show that vector mobilization can be prevented by antiretroviral drugs such as AZT. In contrast to recently published reports by Bukovsky et al. and An et al., the lentivirus vectors used in these studies had little or no effect on the replication and spread of HIV in transduced cells [Bukovsky et al. (1999). J. Virol. 73, 7087-7092; An et al. (1999). J. Virol. 73, 7671-7677]. Whereas vector spread is a significant concern for most gene therapy applications, in the context of gene therapy for HIV infection it may have beneficial effects.     

The influence of riboflavin photochemistry on plasma coagulation factors

Studies with riboflavin in the 1960s showed that it could be effective at inactivating pathogens when exposed to light. The principal mode of action is through electron transfer reactions, most importantly in nucleic acids. This suggested that it could act as a photosensitizer useful in the inactivation of pathogens found in blood products.

Objective

To study the influence of photo-inactivation with riboflavin on the coagulation factors of plasma.

Methods

The photo-inactivation procedure of riboflavin plus light was applied. Fifty isogroup pools of two plasmas were made from 100 U of plasma that were derived from whole blood products that had previously been held overnight. Pools were split into two bags. One of them was photo-inactivated, and post inactivation samples were obtained. The second bag was not photo-inactivated and samples were taken. Total protein, fibrinogen, FII, FV, FVII, FVIII, FIX, FX, FXI, FXIII, antithrombin III, PC, PS, α-2 antiplasmin and vWF:Ag, the multimeric structure of vWF and ADAMTS-13 were analyzed.

Results

In plasma, the proteins most sensitive to photo-inactivation were fibrinogen, FXI, FVIII, FV, and FIX (33%, 32%, 30%, 18% and 18% loss, respectively). Coagulation inhibitors, PS, antithrombin III and PC showed little decrease (all 2%). Retention of vWF and ADAMTS-13 were 99% and 88%, respectively.

Conclusions

As with other pathogen reduction procedures for plasma products, treatment with riboflavin and UV light resulted in reduction in the activity levels of several pro-coagulant factors. Coagulation inhibitors are well preserved.     

Synergistic anti-viral effect of oxanosine and ddI against human immunodeficiency virus.

The combined effect against human immunodeficiency virus (HIV) of oxanosine and 2'3'-dideoxyinosine (ddI) has been evaluated by the production of viral particles, the expression of viral antigens on cell surfaces, and the amount of viral genome integrated in the host cells. Oxanosine alone has no effect on HIV replication up to 100 microg/ml, however, in the presence of ddI, oxanosine revealed concentration dependent inhibition against HIV without cytotoxicity.     

Serum β2-microglobulin in intravenous drug users and its correlation with human immunodeficiency virus infectionin intravenous drug users and its correlation with human immunodeficiency virus infection

Summary High levels of serum β₂-microglobulin have been associated with human immunodeficiency virus infection and β₂-microglobulin has been used with other serological and immunological markers for monitoring disease progression. The usefulness of β₂-microglobulin as a prognostic marker during human immunodeficiency virus infection has been demonstrated in homosexual men and hemophiliacs; few and contradictory data have been reported in intravenous drug users. We have evaluated a cohort of 160 intravenous drug users (81 seronegative and 79 seropositive for human immunodeficiency virus infection) with normal renal function to assess whether serum β₂-microglobulin could be used as a serological marker for monitoring infection; 78 healthy subjects were used as controls. Of 79 seropositive drug users, 54 were asymptomatic or had persistent generalized lymphoadenopathy the remaining 25 had the acquired immunodeficiency syndrome. Seropositive patients were tested for CD4⁺ lymphocyte number, p24 antigen and anti-p24 antibodies. A significant statistical difference was found in mean serum β₂-microglobulin levels between seronegative and seropositive drug users. Moreover, higher levels of β₂-microglobulin were observed in acquired immunodeficiency syndrome patients compared with asymptomatic or patients with persistent lymphadenopathy. A significant relationship was also observed between increased concentration of β₂-microglobulin and the serological and immunological markers which indicate human immunodeficiency virus disease progression.