Keywords: Keywords: W. Lyman, et l, AIDS, Electric Current, lymphoblastoid cell lines (H9 and CEM-SS), HIV-1, AIDS, tritiated thymidine (³H-TdR), HBWSS, Syncytium-formation assay, varicella, The RF Strain, (SDH), H+(ATP).

see also :

http://www.rexresearch.com/kaali/kaali.htm  CLICKHERE   OR HERE   

http://educate-yourself.org/be/ CLICKHERE     OR HERE   

US Patent #  5,185,086  Kaali , et al. February 9, 1993
Method and system for treatment of blood and/or other body fluids and/or synthetic fluids using combined filter elements and electric field forces

US Patent #  5,188,738  Kaali , et al. February 23, 1993
Alternating current supplied electrically conductive method and system for treatment of blood and/or other body fluids and/or synthetic fluids with electric forces

US Patent #  5,139,684   Kaali , et al. August 18, 1992
Electrically conductive methods and systems for treatment of blood and other body fluids and/or synthetic fluids with electric forces

US Patent #  5,817,142  Corder October 6, 1998
Electrical apparatus for killing micro-organisms in the human body

US Patent #  6,539,252  Fields , et al. March 25, 2003
Method and apparatus for the treatment of blood borne pathogens such as immunodeficiency virus

US Patent #  5,352,192  Byrne , et al. October 4, 1994
Medical device

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VIDEO  http://www.youtube.com/watch?v=l_z9FQxfeT0   IS NO LONGER AVAILABLE IN  "YOU TUBE" .....CLICKHERE

For the saved page from youtube  CLICKHERE

For the saved page's title from google  CLICKHERE

SOME STATISTICAL DATA FOR THIS VIDEO EXIST IN  "YOU TOMB" :  http://youtomb.mit.edu/youtube/l_z9FQxfeT0 clickhere

CLICK HERE FOR THE  SAVED WEBPAGE FROM  "YOU TOMB"  

Below follow three ''YOU TUBE" videos with the same theme ........

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http://www.youtube.com/watch?v=XAXCwFJVeuA  

v=XAXCwFJVeuA  for the saved webpage click here

http://www.youtube.com/comment_servlet?all_comments&v=XAXCwFJVeuA&fromurl=/watch%3Fv%3DXAXCwFJVeuA

saved webpage with All Comments (162 total)

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http://www.youtube.com/watch?v=V1otPs4slq0   

v=V1otPs4slq0   for the saved webpage click here

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saved webpage with All Comments (239 total) click here

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http://www.youtube.com/watch?v=kZ4nOCJsbQo 

 v=V1otPs4slq0 for the saved webpage click here

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saved webpage with All Comments ( 37  total) click here

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see also :

http://video.google.com/videoplay?docid=-234247273689402090&hl=en 

http://video.google.com/videoplay?docid=2095786730805958061&hl=en

http://video.google.com/videoplay?docid=-3383948315844437935&hl=en

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http://health.groups.yahoo.com/group/microelectricitygermkiller/

http://health.groups.yahoo.com/group/Beck-n-stuff/

http://bioelectricbuzz.tribe.net/thread/56c3a283-a83c-43ae-a80b-d71704ba5aae

http://www.in-my-opinion.org/in-my-opinion-5016.html&sid=0449e90f9ec8b93aacb5408932db8a9f 

http://community.freespeech.org/blood_electrification_suppressed

http://www.ehealthforum.com/health/topic28850.html

http://curezone.com/forums/am.asp?i=59058

http://www.epanorama.net/wwwboard/messages/8852.html

 ......... Back in 1995 in a 73 magazine editorial I explained about Drs. Lyman and Kaali at the Albert Einstein College of Medicine at Yeshiva University in New York City serendipitously discovering that when a tiny electrical current was passed through the blood it prevented any virus, germs, or parasites trying to live in the blood from replicating, thus killing them ......... For Wayne Green's editorials - referring to Drs. Lyman and Kaali in 10/22/08 - among many other interesting things CLICK HERE http://www.waynegreen.com/wayne/news-archive_2008.html

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http://www.hivwave.gr/pages/en/

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http://www.docgeorge.com/20080521174/Curing-Viruses-With-Electricity.html 

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Inventor:  ZENG XINAN [CN] ; FU XIONG
Applicant:  UNIV SOUTH CHINA TECH
2005-06-08
Inventor(s):  ZENG XINAN [CN]; FU XIONG [CN]; YU SHUJUAN [CN]
Applicant(s):  UNIV SOUTH CHINA TECH [CN]
Classification:  - international:  A23L3/00; A23L3/32; A23L3/00; A23L3/32; (IPC1-7): A23L3/32; A23L3/00
Also published as:  CN1285291  (C)

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Inventor:  ZENG XIN AN [CN] ; LI GUOJI
Applicant:  HUA NAN UNIV OF SCIENCE & ENGI
2003-04-23
Also published as:  CN1194765  (C)

Abstract --  The present invention relates to a treatment device of equipment for sterilizing liquid products in the fields of food, biological, pharmaceutical and chemical industries by adopting high-intensity pulse electric field. Said treatment device is formed from material-treating cavity, two electrodes mounted in the material-treating cavity, inlet and outlet, in which said material-treating cavity is vacuum cavity, and can obtain good sterilizing effect.

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Inventor:  ZENG XIN AN [CN] ; FU XIONG
Applicant:  HUA NAN UNIV OF SCIENCE & ENGI
2002-06-19
Also published as:  CN1174691  (C)

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"Engineering Aspects of Pulsed Electric Field Pasteurization," Zhang, Qinghua, et al., Journal of Food Engineering, 25:261-281, 1994. .

"Inactivation of E. coli and S. cerevisiae by Pulsed Electric Fields Under Controlled Temperature Conditions," Zhang, Q., et al., 1994 American Society of Agricultural Engineers, vol. 37(2);581-587. .

"Inactivation of Microorganisms in a Semisolid Model Food Using High Voltage Pulsed Electric Fields," Zhang, Qinghua, et al., Food Science & Technology (lwt), 1994, 2(6):538..

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1998-04-09
Inventor(s):  YIN YONGGUANG; ZHANG QINGHUA HOWARD; SASTRY SUDHIR KARTIKEYA
Applicant(s):  OHIO STATE RES FOUND
Classification:  - international:  A23L3/00; A23L3/26; A23L3/32; A23L3/00; A23L3/26; A23L3/32; (IPC1-7): A23L3/00; A23L3/26; A23L3/32 - European:  A23L3/00; A23L3/26; A23L3/32
Cited documents:  US4723483 (A)  US4838154 (A)  US5235905 (A)

Abstract --  A pulsed electric field treatment device for the sterilization and preservation of pumpable food products having at least two electrodes (201, 203) and an insulator (202) and particularly suited for the inactivation of vegetative and bacterial spore micro-organisms. Each electrode includes an electrode flow chamber (207, 208) for making electrical contact with the pumpable food product and for allowing the pumpable food product to flow through the treatment devices. The insulator (202) is situated between the electrodes (201, 203) and includes an insulator flow chamber (206) positioned between the electrode flow chambers (207, 208) and provides for the flow of pumpable food product from one electrode flow chamber to the other. A high voltage pulse generator (107) applies a high voltage signal of variable voltage, frequency and pulse duration to the electrodes.; The electrode and insulator flow chambers may employ a variety of sectional and cross-sectional geometries including tubular, cylindrical, rectangular, elliptical and non-uniform design.

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Biotechnol Bioeng. 1992 Dec 20;40(11):1412-20.
Kinetics of sterilization of Lactobacillus brevis cells by the application of high voltage pulses.
Jayaram S, Castle GS, Margaritis A.

The technique of irreversible electroporation has been successfully applied to cause a lethal effect on Lactobacillus
brevis cells suspended in phosphate buffer solution, Na(2)HPO(4)/NaH(2)PO(4) . H(2)O (0.845/0.186 mM) between parallel plane
electrodes. Tests were carried out at different temperatures (24,45,60, and 80 degrees C) to determine if there was a
synergistic effect of temperature and electric pulse treatment on the destruction of L. brevis. Experimental results indicate
that the viability (log N/N(0); where N(0) and N are the number of cells survived per milliliter before and after pulse
voltage application, respectively) of L. brevis decreased with electric field strength E and temperature T and treatment time
t(t). The relations between log(N/N(0)) and t(t) and log(N/N(0)) and E indicate that higher field strengths are more
effective than higher treatment times in causing destruction of L. brevis cells. It was also found that as the temperature of
the liquid medium containing L. brevis cells increased from 24 to 60 degrees C, the death rate of L. brevis cells increased
with a decrease in the total treatment time t(t) (pulse width x number of pulses applied). The application of an electric
field strength E = 25 kV/cm at 60 degrees C and treatment time t(t) = 10 ms resulted in very high destruction levels of L.
brevis cells (N/N(0) = 10(-9)). In comparison with existing steam sterilization technology, this new method of sterilization
using relatively low temperature and short treatment time could prove to be an excellent method to minimize thermal
denaturation of important nutrient components in liquid media. (c) John Wiley & Sons, Inc.

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Appl Microbiol Biotechnol. 1996 Mar;45(1-2):148-57.
Killing of microorganisms by pulsed electric fields.
Grahl T, Märkl H.

Lethal effects of pulsed electric fields (PEF) on suspensions of various bacteria, yeast, and spores in buffer solutions
and liquid foodstuffs were examined. Living-cell counts of vegetative cell types were reduced by PEF treatment by up to more
than four orders of magnitude (> 99.99%). On the other hand, endo- and ascospores were not inactivated or killed to any great
extent. The killing of vegetative cell types depends on the electrical field strength of the pulses and on the treatment time
(the product of the pulse number and the decay time constant of the pulses). For each cell type, a specific critical electric
field strength (Ec) and a specific critical treatment time (tc) were determined. Above these critical values, the fractions
of surviving cells were reduced drastically. The "limits" Ec and tc depend on the cell characteristics as well as on the type
of medium in which the cells are suspended. Especially in acid media living-cell counts were sufficiently decreased at very
low energy inputs. In addition to the inactivation of microorganisms, the effect of PEF on food components such as whey
proteins, enzymes and vitamins, and on the taste of foodstuffs was studied. The degree of destruction of these food
components by PEF was very low or negligible. Moreover, no significant deterioration of the taste of foodstuffs was detected
after PEF treatment. Disintegration of cells by PEF treatment in order to harvest intracellular products was also studied.
Yeast cells, suspended in buffer solution, were not disintegrated by electric pulses. Hence, PEF treatment is an excellent
process for inactivation of microorganisms in acid and in thermosensive media, but not for complete disintegration of
microbial cells.

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Crit Rev Food Sci Nutr. 1996 Jul;36(6):603-27.
Nonthermal pasteurization of liquid foods using high-intensity pulsed electric fields.
Qin BL, Pothakamury UR, Barbosa-Cánovas GV, Swanson BG.

Processing foods with high-intensity pulsed electric fields (PEF) is a new technology to inactivate microorganisms and
enzymes with only a small increase in food temperature. The appearance and quality of fresh foods are not altered by the
application of PEF, while microbial inactivation is caused by irreversible pore formation and destruction of the
semipermeable barrier of the cell membrane. High-intensity PEF provides an excellent alternative to conventional thermal
methods, where the inactivation of the microorganisms implies the loss of valuable nutrients and sensory attributes. This
article presents recent advances in the PEF technology, including microbial and enzyme inactivation, generation of pulsed
high voltage, processing chambers, and batch and continuous systems, as well as the theory and its application to food
pasteurization. PEF technology has the potential to improve economical and efficient use of energy, as well as provide
consumers with minimally processed, microbiologically safe, nutritious and freshlike food products.

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J Food Prot. 1999 Sep;62(9):1088-96.
Pulsed electric field processing of foods: a review.
Jeyamkondan S, Jayas DS, Holley RA.

Use of pulsed electric fields (PEFs) for inactivation of microorganisms is one of the more promising nonthermal
processing methods. Inactivation of microorganisms exposed to high-voltage PEFs is related to the electromechanical
instability of the cell membrane. Electric field strength and treatment time are the two most important factors involved in
PEF processing. Encouraging results are reported at the laboratory level, but scaling up to the industrial level escalates
the cost of the command charging power supply and of the high-speed electrical switch. In this paper, we critically review
the results of earlier experimental studies on PEFs and we suggest the future work that is required in this field.
Inactivation tests in viscous foods and in liquid food containing particulates must be conducted. A successful continuous PEF
processing system for industrial applications has yet to be designed. The high initial cost of setting up the PEF processing
system is the major obstacle confronting those who would encourage the system's industrial application. Innovative
developments in high-voltage pulse technology will reduce the cost of pulse generation and will make PEF processing
competitive with thermal-processing methods.

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Int J Food Microbiol. 2000 Mar 10;54(1-2):91-8.
Pulsed electric fields inactivation of attached and free-living Escherichia coli and Listeria innocua under several conditions.
Dutreux N, Notermans S, Wijtzes T, Góngora-Nieto MM, Barbosa-Cánovas GV, Swanson BG.

The use of pulsed electric fields (PEF) is considered as a mild process in the inactivation of microorganisms present in
liquid food products. PEF treatments of Escherichia coli and Listeria innocua suspended in milk and phosphate buffer, with
same pH and same conductivities, yielded to similar inactivation. Reduction rates obtained in distilled water indicated that
conductivity of the food product is a main parameter in bacterial inactivation. Bacteria attached to polystyrene beads were
inactivated by PEF at a greater (E. coli) or equal rate (L. innocua) than free-living bacteria. Base on the use of selective
and non-selective enumeration media, no clear indications were obtained for sublethal damage of microorganisms surviving the
PEF treatment. E. coli cells subjected to 60 pulses at 41 kV/cm were examined by transmission and scanning electron
microscopy. Changes in the cytoplasm were observed and the cell surface appeared rough. The cells outer membranes were
partially destroyed allowing leaking of cell cytoplasm.

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Radiat Environ Biophys. 1981;20(1):53-65.
Killing of bacteria with electric pulses of high field strength.
Hülsheger H, Potel J, Niemann EG.

Bacteria of the type E. coli K12 have been treated in experiments using high-voltage pulses of short time (microseconds)
as a killing agent. The role of different experimental parameters has been studied: kind of electrolyte, concentration,
length of pulses, field strength, pH and temperature. Electrolytes with bivalent cations were found to reduce the lethal
action. the relative rate of killed bacteria was shown to be mainly governed by the field strength and the treatment time,
which is defined by the product of pulse number and decay time constant. From the obtained results a function has been
developed which enables the precalculation of the killing rate for E. coli, provided that certain limits of experimental
conditions are considered. No correlation between the applied electric energy and the lethal effect could be found.

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Radiat Environ Biophys. 1980;18(4):281-8.

Lethal effects of high-voltage pulses on E. coli K12.

Hülsheger H, Niemann EG.

The lethal effects of high-voltage capacitor-discharges in suspensions of E. coli K12 with varying electrolytes have been examined. A reduction of more than 99.9% of living cells, dependent on the applied voltage could be proved. The bactericidal action is assumed to be due to direct effects of high electric fields. Electrolytically produced chlorine was shown to act as an additional toxic agent, when chloride is present in the treated medium. The relative survival rate of bacteria has been found to depend also on the concentration of cells during pulse treatment.

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Appl Microbiol Biotechnol. 1996 Mar;45(1-2):148-57.

Killing of microorganisms by pulsed electric fields.

Grahl T, Märkl H.

Technische Universität Hamburg-Harburg, Germany.

Lethal effects of pulsed electric fields (PEF) on suspensions of various bacteria, yeast, and spores in buffer solutions and liquid foodstuffs were examined. Living-cell counts of vegetative cell types were reduced by PEF treatment by up to more than four orders of magnitude (> 99.99%). On the other hand, endo- and ascospores were not inactivated or killed to any great extent. The killing of vegetative cell types depends on the electrical field strength of the pulses and on the treatment time (the product of the pulse number and the decay time constant of the pulses). For each cell type, a specific critical electric field strength (Ec) and a specific critical treatment time (tc) were determined. Above these critical values, the fractions of surviving cells were reduced drastically. The "limits" Ec and tc depend on the cell characteristics as well as on the type of medium in which the cells are suspended. Especially in acid media living-cell counts were sufficiently decreased at very low energy inputs. In addition to the inactivation of microorganisms, the effect of PEF on food components such as whey proteins, enzymes and vitamins, and on the taste of foodstuffs was studied. The degree of destruction of these food components by PEF was very low or negligible. Moreover, no significant deterioration of the taste of foodstuffs was detected after PEF treatment. Disintegration of cells by PEF treatment in order to harvest intracellular products was also studied. Yeast cells, suspended in buffer solution, were not disintegrated by electric pulses. Hence, PEF treatment is an excellent process for inactivation of microorganisms in acid and in thermosensive media, but not for complete disintegration of microbial cells.

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J Appl Microbiol. 2003;94(4):571-9.

Modelling and optimization of inactivation of Lactobacillus plantarum by pulsed electric field treatment.

Abram F, Smelt JP, Bos R, Wouters PC.

Food Processing Group, Unilever Research & Development Vlaardingen, The Netherlands.

AIMS: The effect of critical pulsed electric field (PEF) process parameters, such as electric field strength, pulse length and number of pulses, on inactivation of Lactobacillus plantarum was investigated. METHODS AND RESULTS: Experiments were performed in a pH 4.5 sodium phosphate buffer having a conductivity of 0.1 S m-1, using a laboratory-scale continuous PEF apparatus with a co-linear treatment chamber. An inactivation model was developed as a function of field strength, pulse length and number of pulses. Based on this inactivation model, the conditions for a PEF treatment were optimized with respect to the minimum energy required to obtain a certain level of inactivation. It was shown that the least efficient process parameter in the range investigated was the number of pulses. The most efficient way to optimize inactivation of Lact. plantarum was to increase the field strength up to 25.7 kV cm-1, at the shortest pulse length investigated, 0.85 micros, and using a minimum number of pulses. The highest inactivation of Lact. plantarum at the lowest energy costs is obtained by using the equation: E=26.7tau0.23, in which E is the field strength and tau the pulse length. An optimum is reached by substituting tau with 5.1. CONCLUSIONS: This study demonstrates that the correct choice of parameters, as predicted by the model described here, can considerably improve the PEF process. SIGNIFICANCE AND IMPACT OF THE STUDY: The knowledge gained in this study improves the understanding of the limitations and opportunities of the PEF process. Consequently, the advantage of the PEF process as a new option for non-thermal decontamination can be better utilized.

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J Food Prot. 2001 Jul;64(7):964-9.

Reduction in levels of Escherichia coli O157:H7 in apple cider by pulsed electric fields.

Iu J, Mittal GS, Griffiths MW.

Department of Food Science, University of Guelph, Ontario, Canada.

Many studies have demonstrated that high voltage pulsed electric field (PEF) treatment has lethal effects on microorganisms including Escherichia coli O157:H7; however, the survival of this pathogen through the PEF treatment is not fully understood. Fresh apple cider samples inoculated with E. coli O157:H7 strain EC920026 were treated with 10, 20, and 30 instant charge reversal pulses at electric field strengths of 60, 70, and 80 kV/cm, at 20, 30, and 42 degrees C. To accurately evaluate the lethality of apple cider processing steps, counts were determined on tryptic soy agar (TSA) and sorbitol MacConkey agar (SMA) to estimate the number of injured and uninjured E. coli O157:H7 cells after PEF treatment. Cell death increased significantly with increased temperatures and electric field strengths. A maximum of 5.35-log10 CFU/ml (P < 0.05) reduction in cell population was achieved in samples treated with 30 pulses and 80 kV/cm at 42 degrees C. Cell injury measured by the difference between TSA and SMA counts was found to be insignificant (P > 0.05). Under extreme conditions, a 5.91-log10 CFU/ml reduction in cell population was accomplished when treating samples with 10 pulses and 90 kV/cm at 42 degrees C. PEF treatment, when combined with the addition of cinnamon or nisin, triggered cell death, resulting in a reduction in E. coli O157:H7 count of 6 to 8 log10 CFU/ml. Overall, the combination of PEF and heat treatment was demonstrated to be an effective pasteurization technique by sufficiently reducing the number of viable E. coli O157:H7 cells in fresh apple cider to meet U.S. Federal Drug Administration recommendations.

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 J Food Prot. 1999 Jul;62(7):793-6.

Inactivation of Escherichia coli O157:H7 and Escherichia coli 8739 in apple juice by pulsed electric fields.

Evrendilek GA, Zhang QH, Richter ER.

Department of Food Science and Technology, The Ohio State University, Columbus 43210, USA.

The effect of high voltage pulsed electric field (PEF) treatment on Escherichia coli O157:H7 and generic E. coli 8739 in apple juice was investigated. Fresh apple juice samples inoculated with E. coli O157:H7 and E. coli 8739 were treated by PEF with selected parameters including electric field strength, treatment time, and treatment temperature. Samples were exposed to bipolar pulses with electric field strengths of 30, 26, 22, and 18 kV/cm and total treatment times of 172, 144, 115, and 86 micros. A 5-log reduction in both cultures was determined by a standard nonselective medium spread plate laboratory procedure. Treatment temperature was kept below 35 degrees C. Results showed no difference in the sensitivities of E. coli O157:H7 and E. coli 8739 against PEF treatment. PEF is a promising technology for the inactivation of E. coli O157:H7 and E. coli 8739 in apple juice.

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Int J Food Microbiol. 2000 Apr 10;55(1-3):143-6.

Influence of different factors on the inactivation of Salmonella senftenberg by pulsed electric fields.

Alvarez I, Raso J, Palop A, Sala FJ.

Tecnología de los Alimentos, Dpto. PACA, Facultad de Veterinaria, Universidad de Zaragoza, Spain.

The influence of growth phase, cell concentration, pH and conductivity of treatment medium on the inactivation of Salmonella senftenberg by high electric field pulses (HELP) was studied. Cells were more resistant to HELP treatments at the beginning of the logarithmic phase and at the stationary phase. Microbial inactivation was not a function of the initial cell concentration. At constant input voltage, electric field strength obtained in the treatment chamber depended on medium conductivity. At the same electric field strength, conductivity did not influence S. senftenberg inactivation. At the same conductivity, inactivation of S. senftenberg was bigger at neutral than acidic pH.

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J Food Prot. 2003 Jun;66(6):1007-12.

Weibull distribution function based on an empirical mathematical model for inactivation of Escherichia coli by pulsed electric fields.

Rodrigo D, Barbosa-Cánovas GV, Martínez A, Rodrigo M.

Instituto de Agroquímica y Tecnología de Alimentos, Consejo Superior de Investigaciones Científicas, Apartado de Correos 73, 46100 Burjassot, Valencia, Spain.

The pulsed electric field inactivation kinetics of Escherichia coli suspended in orange juices with three different concentrations of carrot juice (0, 20, and 60%) was studied. Electric field strengths ranged from 25 to 40 kV/cm, and treatment times ranged from 40 to 340 micros. Experimental data were fitted to Bigelow, Hülsheger, and Weibull distribution functions, and the Weibull function provided the best fit (with the lowest mean square error). The dependency of each model's kinetic constant on electric field strength and carrot juice concentration was studied. A secondary model was developed to describe the relationship of Weibull parameters a and n to electric field strength and carrot juice concentration. An empirical mathematical model based on the Weibull distribution function, relating the natural logarithm of the survival fraction to treatment time, electric field strength, and carrot juice concentration, was developed. Parameters were estimated by a nonlinear regression. The results of this study indicate that the error rate for the model's predictions was 6.5% and that the model was suitable for describing E. coli inactivation.

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Appl Environ Microbiol. 1999 Dec;65(12):5364-71.

Effects of pulsed electric fields on inactivation kinetics of Listeria innocua.

Wouters PC, Dutreux N, Smelt JP, Lelieveld HL.

Microbiology & Preservation, Unilever Research Vlaardingen, 3133 AT Vlaardingen, The Netherlands. Patrick.Wouters@Unilever.com

The effects of pulsed electric field (PEF) treatment and processing factors on the inactivation kinetics of Listeria innocua NCTC 11289 were investigated by using a pilot plant PEF unit with a flow rate of 200 liters/h. The electric field strength, pulse length, number of pulses, and inlet temperature were the most significant process factors influencing the inactivation kinetics. Product factors (pH and conductivity) also influenced the inactivation kinetics. In phosphate buffer at pH 4.0 and 0.5 S/m at 40 degrees C, a 3. 0-V/microm PEF treatment at an inlet temperature of 40 degrees C resulted in > or = 6.3 log inactivation of strain NCTC 11289 at 49.5 degrees C. A synergistic effect between temperature and PEF inactivation was also observed. The inactivation obtained with PEF was compared to the inactivation obtained with heat. We found that heat inactivation was less effective than PEF inactivation under similar time and temperature conditions. L. innocua cells which were incubated for a prolonged time in the stationary phase were more resistant to the PEF treatment, indicating that the physiological state of the microorganism plays a role in inactivation by PEF. Sublethal injury of cells was observed after PEF treatment, and the injury was more severe when the level of treatment was increased. Overall, our results indicate that it may be possible to use PEF in future applications in order to produce safe products.

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J Food Prot. 1998 Sep;61(9):1203-6.

Inactivation of Listeria monocytogenes in milk by pulsed electric field.

Reina LD, Jin ZT, Zhang QH, Yousef AE.

Department of Food Science and Technology, Ohio State University, Columbus 43210, USA.

Pasteurized whole, 2%, and skim milk were inoculated with Listeria monocytogenes Scott A and treated with high-voltage pulsed electric field (PEF). The effects of milk composition (fat content) and PEF parameters (electric field strength, treatment time, and treatment temperature) on the inactivation of the bacterium were studied. No significant differences were observed in the inactivation of L. monocytogenes Scott A in three types of milk by PEF treatment. With treatment at 25 degrees C, 1- to 3-log reductions of L. monocytogenes were observed. PEF lethal effect was a function of field strength and treatment time. Higher field strength or longer treatment time resulted in a greater reduction of viable cells. A 4-log reduction of the bacterium was obtained by increasing the treatment temperature to 50 degrees C. Results indicate that the use of a high-voltage PEF is a promising technology for inactivation of foodborne pathogens.

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Biotechnol Bioeng. 1992 Dec 20;40(11):1412-20.

Kinetics of sterilization of Lactobacillus brevis cells by the application of high voltage pulses.

Jayaram S, Castle GS, Margaritis A.

Department of Electrical Engineering, University of Western Ontario, London, Ontario, Canada N6A 5B9.

The technique of irreversible electroporation has been successfully applied to cause a lethal effect on Lactobacillus brevis cells suspended in phosphate buffer solution, Na(2)HPO(4)/NaH(2)PO(4) . H(2)O (0.845/0.186 mM) between parallel plane electrodes. Tests were carried out at different temperatures (24,45,60, and 80 degrees C) to determine if there was a synergistic effect of temperature and electric pulse treatment on the destruction of L. brevis. Experimental results indicate that the viability (log N/N(0); where N(0) and N are the number of cells survived per milliliter before and after pulse voltage application, respectively) of L. brevis decreased with electric field strength E and temperature T and treatment time t(t). The relations between log(N/N(0)) and t(t) and log(N/N(0)) and E indicate that higher field strengths are more effective than higher treatment times in causing destruction of L. brevis cells. It was also found that as the temperature of the liquid medium containing L. brevis cells increased from 24 to 60 degrees C, the death rate of L. brevis cells increased with a decrease in the total treatment time t(t) (pulse width x number of pulses applied). The application of an electric field strength E = 25 kV/cm at 60 degrees C and treatment time t(t) = 10 ms resulted in very high destruction levels of L. brevis cells (N/N(0) = 10(-9)). In comparison with existing steam sterilization technology, this new method of sterilization using relatively low temperature and short treatment time could prove to be an excellent method to minimize thermal denaturation of important nutrient components in liquid media. (c) John Wiley & Sons, Inc.

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Int J Food Microbiol. 2004 May 15;93(1):1-10.

Growth of pulsed electric field exposed Escherichia coli in relation to inactivation and environmental factors.

Aronsson K, Borch E, Stenlöf B, Rönner U.

SIK, The Swedish Institute for Food and Biotechnology, Box 5401, 402 29 Göteborg, Sweden.

Pulsed electric fields (PEF) have been proven to inactivate microorganisms during nonthermal conditions and have the potential to replace thermal processing as a method for food preservation. However, there is a need to understand the recovery and growth of survivors and potentially injured microorganisms following PEF processing. The purpose of this investigation was to study the growth of Escherichia coli at 10 degrees C following exposure to electrical field strengths (15, 22.5 and 30 kV/cm) in relation to inactivation and the amount of potentially sublethally injured cells. One medium was used as both a treatment medium and an incubation medium, to study the influence of environmental factors on the inactivation and the growth of the surviving population. The pH (5.0, 6.0 and 7.0) and water activity (1.00, 0.985 and 0.97) of the medium was varied by adding HCl and glycerol, respectively. Growth was followed continuously by measuring the optical density. The time-to-detection (td) and the maximum specific growth rate (micromax) were calculated from these data. Results showed that the PEF process did not cause any obvious sublethal injury to the E. coli cells. The number of survivors was a consequence of the combination of electrical field strength and environmental factors, with pH being the most prominent. Interestingly, the micromax of subsequent growth was influenced by the applied electrical field strength during the process, with an increased micromax at more intense electrical field strengths. In addition, the micromax was also influenced by the pH and water activity. The td, which could theoretically be considered as an increase in shelf life, was found to depend on a complex correlation between electrical field strength, pH and water activity. That could be explained by the fact that the td is a combination of the number of survivors, the recovery of sublethal injured cells and the growth rate of the survivors. Copyright 2003 Elsevier B.V.

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J Appl Microbiol. 2005;99(1):94-104.

Occurrence of sublethal injury after pulsed electric fields depending on the micro-organism, the treatment medium ph and the intensity of the treatment investigated.

García D, Gómez N, Mañas P, Condón S, Raso J, Pagán R.

Departamento de Producción Animal y Ciencia de los Alimentos, Facultad de Veterinaria, Universidad de Zaragoza, Zaragoza, Spain.

AIMS: The objective was to investigate the occurrence of sublethal injury after pulsed electric field (PEF) depending on the treatment time, the electric field strength and the pH of the treatment media in two Gram-positive (Bacillus subtilis ssp. niger, Listeria monocytogenes) and six Gram-negative (Escherichia coli, Escherichia coli O157:H7, Pseudomonas aeruginosa, Salmonella serotype Senftenberg 775W, Salmonella serotype Typhimurium, Yersinia enterocolitica) bacterial strains. METHODS AND RESULTS: A characteristic behaviour was observed for the Gram-positive and Gram-negative bacteria studied. Whereas Gram-positive bacteria showed a higher PEF resistance at pH 7.0, the Gram-negative were more resistant at pH 4.0. In these conditions, in which bacteria showed their maximum resistance, a large proportion of sublethally injured cells were detected. In most cases, the longer the treatment time and the higher the electric field applied, the greater the proportion of sublethally injured cells that were detected. No sublethal injury was detected when Gram-positive bacteria were treated at pH 4.0 and Gram-negative at pH 7.0. CONCLUSIONS: Sublethal injury was detected after PEF so, bacterial inactivation by PEF is not an 'all or nothing' event. SIGNIFICANCE AND IMPACT OF THE STUDY: This work could be useful for improving food preservation by PEF.

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Biochim Biophys Acta. 1996 Jan 12;1278(1):79-88.

Contribution to the biophysics of the lethal effects of electric field on microorganisms.

Kekez MM, Savic P, Johnson BF.

National Research Council of Canada, Ottawa, Canada.

The proposed model assumes that the criteria leading to the lethal breakdown of microorganisms suspended in a continuous medium depend on two parameters: (a) the applied electric field must exceed the critical field of membrane to create holes and (b) the Joule energy (deposited in the membrane) must exceed the minimum value beyond which the cell can not recover. The first parameter initiates (reversible) breakdown and the second one, the completion of the (irreversible) electrical breakdown leading to death of the cell. The number of cells surviving the electric field treatment is related to statistical distribution of cell size. Comparison between theory and the experimental results of Kinosita and Tsong (1977); Hülsheger et al. (1980, 1981, 1983); Rosemberg and Korenstein (1990) and others is given.
PMID: 8611611 [PubMed - indexed for MEDLINE]

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J Water Health. 2004 Dec;2(4):267-77.

The effectiveness of a multi-spark electric discharge system in the destruction of microorganisms in domestic and industrial wastewaters.

Anpilov AM, Barkhudarov EM, Christofi N, Kop'ev VA, Kossyi IA, Taktakishvili MI, Zadiraka YV.

General Physics Institute of the Russian Academy of Sciences, 38 Vavilov Street, Moscow, Russia.

The aim of this work was to investigate the effectiveness of a high voltage multi-spark electric discharge, with pulse energy of 1 Joule, in killing microorganisms in wastewater. Wastewater from primary treated effluent arising from domestic and industrial sources was abstracted for continuous pulsed discharge disinfection. The wastewater contained a large mixed population of microorganisms (approximately 10(7) CFU ml(-1) [10(9) CFU 100 ml(-1)] total aerobic heterotrophic bacteria) including vegetative cells and spores. The electrical conductivity of the wastewater ranged from 900-1400 microS cm(-1) and it was shown that a specific energy of 1.25-1.5 J cm(-3) was required to achieve 1 log reduction in bacterial (faecal coliforms/total aerobic heterotrophs) content. This is higher than that previously shown to reduce the population of E. coli in tap water of low conductivity, demonstrating the role of total wastewater constituents, including dissolved and particulate substances, water colour and the presence of microbial spores, in effective disinfection. The system can be engineered to eradicate microbial populations to levels governed by legislation by increasing treatment time or energy input.

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J Food Prot. 1999 Dec;62(12):1381-6.

High intensity pulsed electric fields applied to egg white: effect on Salmonella Enteritidis inactivation and protein denaturation.

Jeantet R, Baron F, Nau F, Roignant M, Brulé G.

Laboratoire de Technologie Alimentaire, Ecole Nationale Supérieure Agronomique, Rennes, France. jeantet@agrorennes.educagri.fr

High-intensity electric fields have been successfully applied to the destruction of Salmonella Enteritidis in diaultrafiltered egg white. The effects of electric field strength (from 20 to 35 kV x cm(-1)), pulse frequency (from 100 to 900 Hz), pulse number (from 2 to 8), temperature (from 4 to 30 degrees C), pH (from 7 to 9), and inoculum size (from 10(3) to 10(7) CFU x ml(-1)) were tested through a multifactorial experimental design. Experimental results indicate that, for Salmonella inactivation, the electric field intensity is the dominant factor with a strongly positive effect, strengthened by its positive interaction with pulse number. Pulse number, temperature, and pH have also significant positive effects but to a lesser extent. In the most efficient conditions, the pulsed electric field (PEF) treatment is capable of 3.5 log10 reduction in viable salmonellae. Simultaneously, the measure of surface hydrophobicity does not indicate any increase after PEF treatment. These results suggest that no protein denaturation occurs, unlike what is observed after comparable heat treatment in terms of Salmonella inactivation (55 degrees C for 15 min).

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Antimicrob Agents Chemother. 1994 Dec;38(12):2803-9.

Mechanism of electrical enhancement of efficacy of antibiotics in killing biofilm bacteria.

Costerton JW, Ellis B, Lam K, Johnson F, Khoury AE.

Center for Biofilm Engineering Montana State University, Bozeman. 59717-0398.

The bioelectric effect, in which electric fields are used to enhance the efficacy of biocides and antibiotics in killing biofilm bacteria, has been shown to reduce the very high concentrations of these antibacterial agents needed to kill biofilm bacteria to levels very close to those needed to kill planktonic (floating) bacteria of the same species. In this report, we show that biofilm bacteria are readily killed by an antibiotic on all areas of the active electrodes and on the surfaces of conductive elements that lie within the electric field but do not themselves function as electrodes. Considerations of electrode geometry indicate that very low (< 100 microA/cm2) current densities may be effective in this electrical enhancement of antibiotic efficacy against biofilm bacteria, and flow experiments indicate that this bioelectric effect does not appear to depend entirely on the possible local electrochemical generation of antibacterial molecules or ions. These data are expected to facilitate the use of the bioelectric effect in the prevention and treatment of device-related bacterial infections that are caused by bacteria that grow in biofilms and thereby frustrate antibiotic chemotherapy.

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Antimicrob Agents Chemother. 2004 Dec;48(12):4662-4.

A radio frequency electric current enhances antibiotic efficacy against bacterial biofilms.

Caubet R, Pedarros-Caubet F, Chu M, Freye E, de Belém Rodrigues M, Moreau JM, Ellison WJ.

Unité Sécurité Microbiologique des Aliments, Institut des Sciences et Techniques des Aliments de Bordeaux, Université de Bordeaux 1, Talence, France. r.caubet@istab.u-bordeaux1.fr

Bacterial biofilms are notably resistant to antibiotic prophylaxis. The concentration of antibiotic necessary to significantly reduce the number of bacteria in the biofilm matrix can be several hundred times the MIC for the same bacteria in a planktonic phase. It has been observed that the addition of a weak continuous direct electric current to the liquid surrounding the biofilm can dramatically increase the efficacy of the antibiotic. This phenomenon, known as the bioelectric effect, has only been partially elucidated, and it is not certain that the electrical parameters are optimal. We confirm here the bioelectric effect for Escherichia coli biofilms treated with gentamicin and with oxytetracycline, and we report a new bioelectric effect with a radio frequency alternating electric current (10 MHz) instead of the usual direct current. None of the proposed explanations (transport of ions within the biofilm, production of additional biocides by electrolysis, etc.) of the direct current bioelectric effect are applicable to the radio frequency bioelectric effect. We suggest that this new phenomenon may be due to a specific action of the radio frequency electromagnetic field upon the polar parts of the molecules forming the biofilm matrix.

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Lab Chip. 2005 Sep;5(9):943-8. Epub 2005 Jul 26.

A new pulsed electric field microreactor: comparison between the laboratory and microtechnology scale.

Fox M, Esveld E, Luttge R, Boom R.

Food and Bioprocess Engineering Group, Wageningen University, P.O. Box 8129, 6700 EV, Wageningen, The Netherlands. Martijn.Fox@wur.nl

This paper presents a new microreactor dedicated for pulsed electric field treatment (PEF), which is a pasteurization method that inactivates microorganisms with short electric pulses. The PEF microreactor consists of a flow-through channel with a constriction where the electric field is focussed. Compared to a laboratory-scale setup 25 times lower voltages were needed to obtain the same electric field strength due to the close electrode spacing. A finite element model showed that the electric field intensity is very homogeneous throughout the channel, which is crucial for the pasteurization processes. Experiments where artificial vesicles, loaded with carboxyfluorescein, were electroporated showed that the maximum transmembrane potential adequately described the processes both in the microreactor and the laboratory-scale setup, although the length scales are different. Electroporation started at a transmembrane potential of 0.5 V, reaching a maximum fraction of electroporated vesicles of 51% at a transmembrane potential of 1.5 V. The partial electroporation is not a result of the heterogenity of the vesicles or the electric field. With this new PEF microreactor it is possible to study the PEF process in more detail.

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Anticancer Res. 2001 May-Jun;21(3B):1809-15.

A new antitumour treatment combining radiation and electric pulses.

Engström PE, Persson BR, Brun A, Salford LG.

Department of Radiation Physics, Lund University Hospital, Sweden.

AIM: To investigate the antitumour effect of radiation in combination with electropermeabilization on subcutaneous rat glioma tumours. MATERIALS AND METHODS: Sub-optimal radiation treatment was administered separately or in combination with electric pulses of high voltage to subcutaneous rat brain tumours. The treatment was repeated on four consecutive days and evaluated by TGD and microscopical examination. The tumours were stained for Factor VlII/von Willebrand Factor to investigate the effects on the tumour vasculature. RESULTS: Radiation and electric pulses applied concomitantly resulted in a cure rate of 67% (tumour free >80 days after treatment). Radiation-treated animals showed progressive disease. Histological and immunohistochemical examination of electric impulse-treated tumours showed instant and severe deteriorating effects on tumour vasculature. CONCLUSION: A distinct antitumour effect of the combined treatment of electric pulses and radiation treatment was observed. We believe that the tumouricidal effect arises from destruction of the tumour vasculature but also from DNA related damage from reactive oxygen formed by the electric pulses and the radiation treatment.

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Int J Food Microbiol. 2003 Oct 15;87(1-2):87-95.

The influence of process parameters for the inactivation of Listeria monocytogenes by pulsed electric fields.

Alvarez I, Pagán R, Condón S, Raso J.

Tecnología de los Alimentos, Facultad de Veterinaria, Universidad de Zaragoza, 50.013 Saragossa, Spain.

The influence of the electric field strength, the treatment time, the total specific energy and the conductivity of the treatment medium on the Listeria monocytogenes inactivation by pulsed electric fields (PEF) has been investigated. L. monocytogenes inactivation increased with the field strength, treatment time and specific energy. A maximum inactivation of 4.77 log(10) cycles was observed after a treatment of 28 kV/cm, 2000 micros and 3490 kJ/kg. The lethal effect of PEF treatments on L. monocytogenes was not influenced by the conductivity of the treatment medium in a range of 2, 3 and 4 mS/cm when the total specific energy was used as a PEF control parameter. A mathematical model based on the Weibull distribution was fitted to the experimental data when the field strength (15-28 kV/cm), treatment time (0-2000 micros) and specific energy (0-3490 kJ/kg) were used as PEF control parameters. A linear relationship was obtained between the log(10) of the scale factor (b) and the electric field strength when the treatment time and the total specific energy were used to control the process. The total specific energy, in addition to the electric field strength and the treatment time, should be reported in order to evaluate the microbial inactivation by PEF.

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Water Res. 2002 Aug;36(14):3429-38.

Elimination of free-living amoebae in fresh water with pulsed electric fields.

Vernhes MC, Benichou A, Pernin P, Cabanes PA, Teissié J.

Institut de Pharmacologic et de Biologie Structurale, CNRS UMR 5089, Toulouse, France.

This study investigates the effects of pulsed electric fields on the inactivation of trophozoite form of Naegleria lovaniensis Ar9M-1 in batch and flow processes, systematically examining the lethal effect of field strength, pulse duration, number of pulses, and pulse frequency. Our results show that amoebae eradication is modulated by pulse parameters, composition of the pulsing medium, and physiological state of the cells. Cell survival is not related to the energy delivered to the cell suspension during the electrical treatment. For a given energy a strong field applied for a short cumulative pulse duration affects viability more than a weak field with a long cumulative pulsation. We also determine the optimal electrical conditions to obtain an inactivation rate higher than 95% while using the least energy. Flow processes allow to treat large-scale volumes. Our results show that the most efficient flow process for amoeba eradication requires a field parallel to the flow. Pulsed electric fields are a new and attractive method for inactivating amoebae in large volumes of fresh water.

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J Dairy Sci. 2006 Mar;89(3):905-11.

Comparative study on shelf life of whole milk processed by high-intensity pulsed electric field or heat treatment.

Odriozola-Serrano I, Bendicho-Porta S, Martín-Belloso O.

Department of Food Technology UTPV-CeRTA, University of Lleida Rovira Roure 191, 25198 Lleida, Spain.

The effect of high-intensity pulsed electric fields (HI-PEF) processing (35.5 kV/cm for 1,000 or 300 micros with bipolar 7-micros pulses at 111 Hz; the temperature outside the chamber was always < 40 degrees C) on microbial shelf life and quality-related parameters of whole milk were investigated and compared with traditional heat pasteurization (75 degrees C for 15 s), and to raw milk during storage at 4 degrees C. A HIPEF treatment of 1,000 micros ensured the microbiological stability of whole milk stored for 5 d under refrigeration. Initial acidity values, pH, and free fatty acid content were not affected by the treatments; and no proteolysis and lipolysis were observed during 1 wk of storage in milk treated by HIPEF for 1,000 micros. The whey proteins (serum albumin, beta-lactoglobulin, and alpha-lactalbumin) in HIPEF-treated milk were retained at 75.5, 79.9, and 60%, respectively, similar to values for milk treated by traditional heat pasteurization.

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Lett Appl Microbiol. 2002;35(1):90-4.

Pulsed high voltage electric discharge disinfection of microbially contaminated liquids.

Anpilov AM, Barkhudarov EM, Christofi N, Kop'ev VA, Kossyi IA, Taktakishvili MI, Zadiraka Y.

General Physics Institute, Moscow, Russia.

AIMS: To examine the use of a novel multielectrode slipping surface discharge (SSD) treatment system, capable of pulsed plasma discharge directly in water, in killing micro-organisms. METHODS AND RESULTS: Potable water containing Escherichia coli and somatic coliphages was treated with pulsed electric discharges generated by the SSD. The SSD system was highly efficient in the microbial disinfection of water with a low energy utilization (eta approximately 10-4 kW h l-1). CONCLUSIONS: The SSD treatment was effective in the destruction of E. coli and its coliphages through the generation of u.v. radiation, ozone and free radicals. SIGNIFICANCE AND IMPACT OF THE STUDY: The non-thermal treatment method can be used for the eradication of micro-organisms in a range of contaminated liquids, including milk, negating the use of pasteurization. The method utilizes multipoint electric discharges capable of treating large volumes of liquid under static and flowing regimes.

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Anal Bioanal Chem. 2004 Nov;380(5-6):831-7.

Simultaneous determination of multiple constituents in real beer samples of different origins by capillary zone electrophoresis.

Cortacero-Ramírez S, Segura-Carretero A, Cruces-Blanco C, Romero-Romero ML, Fernández-Gutiérrez A.

Research Laboratories of Grupo Cervezas Alhambra, S.L. Avda. Murcia 1, 18010 Granada, Spain.

Simultaneous determination of alcohols, amines, amino acids, flavonoids, and purine and pyrimidine bases in bottled beer samples directly without any pre-treatment was carried out by capillary zone electrophoresis with diode-array detection. Electrolyte conditions such as pH, composition and concentration of the buffer, working voltage and type and time of injection were checked. The best separation of the cited analytes was achieved in 70 mM sodium borate solution and pH 10.25. The detection limits were from 2.1 to 5.6 mg L(-1) for the 18 compounds studied. The developed method is rapid, sensitive and quantitative and has been applied to seven types of international bottled beers of different origins bought locally.

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Endod Dent Traumatol. 1985 Jun;1(3):112-5.

Effect of electric current and silver electrodes on oral bacteria.

Tronstad L, Trope M, Hammond BF.

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Zhongguo Zhong Yao Za Zhi. 1992 Oct;17(10):604-6, 639.

Research on sterilization of pathogens by high electrostatic voltage method

Wang X, Wu Y, Ni X, Xia B, Xu J, Du Q.

Institute of Electrostatics, Northeast Normal University, Changchun.

An experimental research has been carried out on the sterilization of four kinds of pathogens by high electrostatic method along with an inquiry into the influence of voltage waveform and the treated time on sterilization. It is concluded that pathogens can be killed efficiently by corona discharge field.

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http://www.ncbi.nlm.nih.gov/pubmed/9151574 

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http://www.ncbi.nlm.nih.gov/pubmed/11588820?dopt=Abstract  

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Biocompatible electric current attenuates HIV infectivity http://www.ncbi.nlm.nih.gov/pubmed/15858720?dopt=Abstract  click here

or here,  for the saved page

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BIOCOMPATIBLE ELECTRIC CURRENT ATTENUATES HIV-I INFECTIVITY

William D. Lyman, Irwin R.Merkatz
William C. Hatch and Steven C. Kaali
Departments of Pathology,
and Obstetrics & Gynecology
Albert Einstein. College of Medicine,
1300 Morris Park Ave., Bronx, N.Y.10461

Running title: Electricity reduces HIV-1 infectivity

Correspondence: Dr, Wm.. D. Lyman
Department of Pathology
Albert Einstein College of Medicine
1300 Morris Park Avenue
The Bronx, NY 10461
(212) 430-2171

SUMMARY

In this report, we present the results of double-blinded studies on the use of direct electric current to alter the infectivity o£ HIV-1 for susceptible cells in vitro. Two lymphoblastoid cell lines (H9 and CEM-SS) were exposed to aliquots of the RT strain of HIV-1 treated with direct current. Results of these studies show that virus treated with currents from 50 to 100 microamperes (ìA) has a significantly reduced infectivity for susceptible cells.

These experimental currents were equal to 3.85 and 7.7.ìÁ/mm² current densities respectively. The reduction of infectivity was dependent upon, the total electric charge (ìA x min) passing through the chamber to which the virus was exposed. Viral infectivity was determined by two independent measures: a syncytium-formation assay which can be used to quantify the production of infectious particles; and. a reverse transcriptase assay which is an index of viral protein production. Additional experiments demonstrated that the currents employed were biocompatible. Uninfected H9 cells were exposed to the same conditions used for the viral aliquots.

There was no significant change in the percentage of viable uninfected cells exposed to any of the currents tested. Therefore, because biocompatible direct electric current attenuates the infectivity of cell-free virus, this treatment may allow development of new strategies to prevent transmission of HIV-1 through either treating the general blood supply or developing alternative barrier contraceptive devices. Additionally, biocompatible electric. current may be applicable for the direct treatment of AIDS patients by utilizing either extracorporeal systems or self contained indwelling electrodes. Lastly, because the virus is being attenuated, electric current may also render treated HIV-1 suitable for vaccine development.

Key words: HIV-1, AIDS, treatment, suppression of infectivity, electricity

INTRODUCTION

The number of individuals infected by the human immunodeficiency virus type-1 (I-(HIV-1) continues to increase on a world-wide basis (1). A significant percentage, if not all, of these individuals will eventually develop the acquire d immunodeficiency syndrome (AIDS) (2)- While horizontal transmission in the homosexual. population may be contained or decreasing (3), heterosexual transmission and infection through contaminated blood supplies continues to increase (4). Additionally ver tical transmission from infected females to their fetuses is also on the rise with a resultant increase in the number of children with AIDS (5). New strategies, therefore, must be devised in order to limit more effectively the spread of this virus.

In this regard, three principal approaches are currently being investigated. In order to decrease susceptibility to the consequences of infection, vaccines are being sought which will induce the production of protective antibodies (6). As treatment modalities, the use of soluble antagonists to block the receptor for HIV-1 is being studied (7) as are pharmacologic agents such as nucleic acid analogs which can interfere with the transcription of viral genomic sequences (8). Each of these systems has------------ and limitations and to date none has proven completely effective.

Because heat or light in combination with drugs and dyes can inactivate viruses including HIV-2 in vitro (9), others have suggested the use of these forms of energy to treat .. AIDS patients. The results of studies using heat have not been peer- reviewed and are therefore impossible to evaluate. The use of light with drugs ["photopheresis"] (10) appears to be efficacious although this treatment may be limited by drug toxicity and the potential long-term effects of ultraviolet radiation on blood c ell nucleic acids. Also, by its nature, this last system may not be suitable for the treatment of tissue-associated virus.

As result of our interest in the use of electric current to alter biological systems , we focused our investigations on the ability of direct electrical current at biocompatible levels to alter the infectivity of HTV-1 for susceptible CD4 positive cell s in vitro.

MATERIALS AND METHODS

Electrical treatment of HIV 1:

The RF strain of HIV-I (AIDS Reagent Program) was cryopreserved prior to treatment at -70°C. Fur treatment, a sample of virus was thawed and maintained on ice at 4°C . Ten microliters (ìl) of HIV-1 at a concentration of 105 infectious particles per ml were placed into a chamber which included a pair of platinum electrodes 1mm apart permanently mounted into a well 1.56mm in length an d 8.32mm in depth equal to 12.9 ìl volume capacity. The chamber was connected to a power supply capable of creating constant direct current. The viral aliquots were exposed to direct currents ranging from 0 microamperes ( ìA) for up to 12 minutes to 100ìA for up to 6 minutes. Intermediate currents of 25, 50 and 75ìA were used to expose similar viral aliquots. Under these conditions, for example, 0, 50 and 100ìA represent 0, 3.85 and 7 .7ìA/mm² current densities respectively. The current was monitored throughout the experiment. A matrix of current and time employed is shown in Table 1.

After the exposure of virus to electric current, the contents of the chamber were removed and placed into sterile microtubes. Five ìl of each sample were removed and diluted with 95ìl tissue culture medium supplemented with 10% fetal calf serum (FCS) for subsequent assays.

Syncytium-formation assays:

This assay was performed as previously described by Nara et al (11). Briefly, 10⁵ CEM-SS cells were dispensed into poly-L-lysine coated microliter wells. Thereafter, tenfold dilutions o f H9 cells incubated with the treated HIV-1 samples were co-cultured in triplicate for up to 4 days with the CEM-SS cells. Identical wells were prepared with control uninfected and infected cells. The wells were examined for syncytium formation at 2 and 3 days and quantified using an inverted microscope.

RReverse trascriptase assay:

Uninfected H9 cells, were pelleted at 1,000 rpm for minutes at room temperature, the supernatant was decanted and the cells were resuspended in 100ìl treated viral sample. The cells were incubated for up to 6 hours with the viral samples. At the end of the incubation time, the viral/cell suspensions were centrifuged at 1,000 RPM for 5 minutes and the supernatant decanted. The cell pellet was then resuspended in 5ml of RPMI, 10% FCS and placed into a T25 tissue culture flask and maintained at 37⁰C, 5% CO₂ in a humidified chamber. At 2 day intervals (beginning at day 2}, 1ml of the cell suspensions was removed from each sample and centrifuged at 1,000 rpm for 5 minutes in order to pellet the cells. The supernatant was subsequently centrifuged at 14,000 RPM for I5 minutes. The pellet was resuspended in suspension buffer and assayed using standard methodology employing Mg+ + as the divalent cation poly (rA) oligo d(T) 12-18 as template primer, and tritiated thymidine (³H-TdR) which comprise the reaction mixture. Known HIV positive and negative control samples were included in each assay for reference. Thirty ìl of the reacti on mixture were added to each 10 ìl viral sample and incubated at 37 ⁰C for 60 min. Samples were then incubated with 1ml of cold quench solution on ice for 15 minutes and filtered through a Millipore manifold. Chimneys were rinsed first with wash solution and followed by cold 95% ethanol. The filters were dried by vacuum and counted in scintillation fluid. Reverse transcriptase activity is expressed as counts per minute (cpm) and is considered positive only if cpm are at least five times greater than the cpm obtained with HIV negative control samples.

Biocompatibility of electric currents/time:

To determine if the electric currents used were in a biocompatibility range of energy, uninfected H9 cells were exposed to distinct currents for different amounts of time. The H9 cells were washed two times in Hanks Balance Salt Solution (HBSS). Thereafter, the cells were resuspended in RPMI, 10% FCS at a concentration of 10⁶ cells per ml, Ten ìl of the cell samples were placed into the reaction c hamber. The cell samples were then exposed to 0, 50 or 100ìA for 0, 3 or 6 minutes. At the end of each test, the cell sample was removed from the chamber and approximately 10ìl of the sample was mixed with 90ìl of trypan blue. The number of viable cells w as determined by trypan blue exclusion using a hemocytometer and tight microscope. Results are expressed as percentage of viable cells from the total of all cells. At least 200 cells per field were counted.

Statistical analysis:

Results of the syncytium-formation and reverse transcriptase assays were tested for statistical significance by the Student's t test and analyses of variance.

RESULTS

Syncytium-formation assay:

Using this index of HIV-1 infectivity, it was determined that exposing virus to direct electric current suppressed its capacity to induce the formation of syncytia. Figure 1 shows a representative e xperiment and Table 2 shows the Croup data for 3 separate experiments. As can be noted in Figure l, a statistically significant (p<0.001) reduction in sycytium number was observed and this reduction was dependent upon the current applied to the viral i solate. At three different viral dilutions, there were analogous results in that a total charge of 200ìA x min (25ìA for 8 minutes) reduced the number of syncytia from 50 to 65% while a charge of 300ìA x min (50ìA for 6 minute s, 75ìA for 4 minutes or 100ìA for 3 minutes) resulted in 90% reduction.

Reverse transcriptase assays:

The direct electric currents to which HIV-1 was exposed also reduced reverse transcriptase activity. Five separate experiments were conducted and a representative experiment is shown in Figure 2 and the ;coup data are included in Table 3. As can be seen in Figure 2, there was a significant decrease in the amount of reverse transcriptase activity after exposure of the virus to either 50ìA for 3 or 6 minutes. An equivalent reduction in reverse transcriptase activity was also noted with exposure to, 100ìA for 3 minutes and almost ablation of reverse transcriptase activity was seen with exposure of the viral isolate to 100ìA for 6 minutes. The group data (Table 3} show that after exposure to 50ìA for 6 minutes, there was a 44% reduction in activity and treatment of virus with 100ìA for 6 minutes resulted in a 94% reduction. An analysis of variance indicates that t he decrease in reverse transcriptase activity was statistically significant (p <0.0001).

Biocompatibility of the electric currents/time:

The results of a viability analysis using trypan blue exclusion criteria applied to uninfected cells exposed to the different currents and times used far these studies are shown in Table 4. The viability of H9 cells, after exposure to 100ìA fur either 3 or b minutes, did not show a significant decrease when compared to the 0 Current control. After maximum treatment at 100ìA for 6 minutes, cell viability was 93%. Interestingly, in other preliminary experiments in which HIV-infected H9 cells were used, the results show that at 100 ìA there may have been a significant decrease in the number of viable cells. That is, while an insta ntaneous pulse of 100 ìA did not affect the viability of infected cells, at 3 and 6 minutes of exposure to 100 ìA, a decrease in viability was noted. This decrease was time dependent in that exposure to 100 ìA far 3 min utes resulted in a viability of 83% while 100 ìA for 6 minutes resulted in a viability of 80%. Although these data are provocative, they only represent a preliminary experiment and require further investigation.

With respect to the possibility that the electric current was transduced into heat, the calculated rise in temperature within the chamber was determined to be less than 1°C. In order to verify this, a temperature microprobe was introduced into the cham ber containing tissue culture medium alone. Results of these studies are shown in Table S. Similar results were obtained when H9 cell-containing medium was placed in the reaction chamber. The data indicate that for the currents and times used for these ex periments, there was no alteration in the temperature of the chamber.

DISCUSSION

The results reported here demonstrate that HIV-1 treated with direct electric currents from 50 to 100ìA has a significantly reduced infectivity for susceptible cells in vitro. This reduction o f infectivity correlates with the total electric change passing through the chamber. Although extrapolation of these data predicts that ablation of HIV infectivity may be possible, and additional preliminary data support this prediction, the expectation t hat some virions may still escape the electrical effect cannot be discounted. Nevertheless, the .therapeutic potential of electric current may reside in its ability to lower the viral titer to subclinical significance or in its incorporation into a strate gy analogous to that of other therapies in which repeated cycles of treatment eventually achieve remission or cure.

The data presented in this report are based on both quantitative and quantal determinations of viral infectivity. Although the syncytium-formation assay can be used to quantify the number of infectious viral particles, this use with respect to HI V-1 may be abridged because of the ability of free fusigenic peptide (gp41) to induce syncytia by itself. Therefore, while syncytia were observed at some dilutions of electrically-treated virus, this may simply represent the presence of soluble gp41 in th e tissue culture medium. We believe that the correlation between total charge and reduction in syncytium number more adequately reflects the ability of direct electric current to reduce HIV-1 infectivity.

This belief is also supported by the results of the reverse transcriptase assays.

Although a decrease in HIV-1 reverse transcriptase does not assure reduced infectiousness of this virus for Susceptible cells; we feel that, taken together with the syncytium-formation data, the results indicate that significant attenua tion of HIV-I infectivity is achieved by treatment with direct electric currents.

With respect to the biocompatibility of the electric currents and total charges reported here, two separate sets of evidence are applicable. The first has to do with the results showing that, by trypan blue exclusion, no significant cyt otoxicity was induced in by any total charge tested. The other evidence is obtained from reports which clearly indicates that the amount of electricity used for these experiments is significantly below presently used therapeutic electric currents which ar e in the milliampere range (12-16).

Rather than negative effects, exposure of cells to electric current may actually have positive consequences for resistance to infection in that important cellular electrochemical changes correlate with enhancement of specific enzymatic activities. In particular, a facilitation of succinate dehydrogenase (SDH) and ATPase activity has been observed (12,15). Both of these enzymes are associated with the oxidative capacity of the cell. Specifically, it has been suggested that an elec trochemical reaction occurs between mitochondrial membrane-bound H+ ATPase and ADP leading to the formation of ATP. Therefore, exposure of cells to direct electric current may directly or indirectly increase energy resources within a cell and facil itate cell metabolism. This, in turn, may actualIy render a cell less susceptible to the effects of viral infection.

In summary, the data presented here indicate that biocompatible direct electric current significantly reduces the infectivity of HIV-1. Continuing investigations are exploring the mechanisms through which this effect is mediated. The in itial focus of these experiments is centered on the potential role which ionic and molecular species generated by electrolysis may have on the virus. However, the complete mechanism by which direct electric current attenuates HIV-1 infectivity is undoubte dly far re complex than simple electrolysis. Nonetheless. and independent of a complete understanding of all of the mechanisms involved in the attenuation of HIV-1 infectivity, the present observations may serve as an initiaI step for the development of new strategies to treat infection or prevent transmission of HIV-1 through either treat ing the general blood supply or developing alternative barrier contraceptive devices. It may also be feasible to treat AIDS patients with direct electric current using either extracorporeal systems or self contained indwelling electrodes. Lastly, because viral infectivity is being attenuated, electric current may render treated HIV-1 suitable for vaccine development.

ACKNOWLEDGMENTS

Thanks go to Mrs. Agnes Geoghan for her excellent secretarial assistance and to Dr.Gabor, Kemeny for important technical help. Additional thanks go to Drs. Frank Lilly and Philip Aisen for their constructive criticism of this manuscript.

LEGENDS

REFERENCES

1. Sato PA, Chin J, Mann JM. Review of AIDS and HIV infection Giobal epidermiology and statistics. AIDS 1989; 3 Suppl.1:S301-7.

2. Centers for Disease Control. Revision of the CDC surveillance case definition for acquired immunodeficiency syndrome. MMWR 1987; 1 Suppl. 36:S1-15.

3. Thacker SB, Berkelman RL. Public health surveillance in the United States. Epidemiol. Rev 1988; 10:164.90.

4. Klein RS, Friedland GH. Transmission of human immunodeficiency virus type (HIV-1) by exposure to blood: Defining the risk. Ann Int Med 1990; 113:729-30.

5. Oxtoby MJ. Epidemiology of pediatric AIDS in the United States. In: Brain in Pediatric AIDS (Kozlowski PB, Snider DA, Vietze PM, Wisniewski HM, eds) 1990:1-8

6. Broder S, Mitsuya H, Yarchoan R, Pavlakis GN. Antiretroviral therapy in AIDS. Ann Int Med 1990: 113:604-18.

7. Perno CF, Baseler MW, Broder S, Yarchoan R. Infection of monocytes by human immunodeficiency virus I blocked by inhibitors of CD4-gp120 binding, even in the presence of enhancing antibodies. J Exp Med 1990; I71:1043-56.

8. Mitsuya H, Weinhold KJ, Furman FA et al. 3'-Azido-3'-deoxythymidine (BW A509U): an antiviral agent that inhibits the infectivity and cytopathic effect of human T-lymphotropic virus type III/ lymphadenopathy-associated virus in vitro Proc Natl Acad Sci USA 1985; 82:7096-100.

9. Quinnan GV, Wells MA, Wittek AE, et al. Inactivation of human T-cell virus, type III by heat, chemicals and irradiation. Transfusion 1986; 26:481-3.

10. Bisaccia E, Berger C, KIainer AS. Extracorporeal photopheresis in the treatment of AIDS-related complex: A pilot study. Ann Int Med 1990; 113:270-75.

11. Nara PL, Hatch WC, Dunlop NM, et al.: Simple, rapid quantitative, syncytium-forming microassay for the detection of human immunodeficiency virus neutralizing antibody. Aids Res Hum Retrovirus 1987; 3:283-302

12. Cheng N, Van Hoof H, Bockx E, et al. The effects of electric currents on ATP generation, protein synthesis, and membrane transport in rat skin. Clin Ortho ReI Res 1982; 17I:26472.

13. Frank G, Schachar N, Dittrich D, et al. Electromagnetic stimulation of ligament healing in rabbits. Clin Ortho ReI Res 1983; I75:263-72.

14. Eriksson E, Haggmark T. Comparison of isometric muscle training and electrical stimulation supplementing isometric muscle training in the recovery after major knee ligament surgery. Amer J Sports Med 19?9; 7:159-71.

15. Stanish WD, Valiant GA, Bonen A, et al. The effects of immobilization and of electrical stimulation on muscle glycogen and myofibrillar ATPase. Can J Appl Sport Sci 1982; 7:267-71.'

16. Pills AA. Electrochemical information transfer at living cell membranes. Ann NY Acad Sci 1974; 205:148-70.

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