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Microsoft word - angola electrotherapy study - 9 march 2006.doc

Investigation of the effect of a multi wave oscillator in the
treatment of adult HIV/AIDS patients:

Authors: Campbell N R, Paspaliaris V, Ballard R

Enquiries: Prof. N. Campbell, P O Box 137, Parkville. Vic. 3052 Australia
Email: noelc@smile.org.au
Abstract
In this study 26 patients infected with the HIV-1 virus were treated daily for
two months with an electrotherapy device and their results compared with
27 non-treated patients matched for sex and age who were not participating
in any anti-HIV treatment.
Over the 2 months of the trial, CD4 cell numbers reduced by 12% in the
non-treated patients.
The treated group showed a 5% increase in CD4 cell numbers.
CD8 cell numbers dropped by 17% in the non-treated patients, and
increased by 18% in the treated patients, over the same 2 months.
In addition HIV viral loads increased by 109% in the untreated patients, and
decreased by 6% in treated patients.
In many parts of the world where HIV infection is particularly rife, there are
insufficient financial resources to pay for treatment with anti-viral drugs.
This electrotherapy technique appears to be a low cost method for treating
HIV infected patients who cannot afford conventional anti-viral treatment,
and so are currently receiving no treatment at all.
Further investigation of this electrotherapy methodology in large-scale
multi-centre trials is warranted.
Introduction

It is well known that human immunodeficiency virus (HIV) infection leads to
depressed cellular immunity, which can result in serious opportunistic infections
in acquired immune deficiency syndrome (AIDS) patients (Gottlieb et al. 1981;
Gallo et al. 1984). At present, combination anti-retroviral drug regimens including
protease inhibitors are used as a standard therapy for HIV-1 infection.
Access to antiretroviral drugs for HIV-infected patients in developing countries is
a global public health priority. With the support of multilateral and bilateral programmes, non-governmental organisations, and national authorities, WHO has the ambitious objective to treat 3 million people with highly active antiretroviral therapy (HAART) by 2005 (WHO, UNAIDS 2003). WHO currently recommends first-line therapy with two nucleoside reverse transcriptase inhibitors (NRTIs) and one non-NRTI (NNRTI), a combination with good efficacy, tolerability and simplicity, low cost, and good adherence to treatment (WHO 2003). Generic fixed-dose combinations of such regimens are widely regarded as crucial for scaling-up AIDS treatment in developing countries. These treatments improve adherence owing to the fewer daily doses relative to individual formulations. Supply, storage, and distribution are also easier because the range of products is smaller. Generic drugs are generally much cheaper than brand-name formulations. Several generic fixed-dose combinations have been pre-qualified by WHO (WHO 2004) after assessment of manufacturers’ product data (including data for purity of all ingredients, stability of the finished products, and results of in-vivo bioequivalence tests), actual pharmacological composition, and manufacturing practices. However, these formulations are not yet recommended by some of the major donor agencies, such as the US government's multi-billion dollar PEPFAR (President's emergency plan for AIDS relief funding) programme for developing countries (USAID 2004). In addition to political considerations, particularly on the legitimacy and consequences of using generic instead of brand-name drugs, this situation is partly explained by the absence of clinical studies showing the efficacy and tolerability of generic fixed-dose combinations. Quality control of different drug batches is also a difficulty in most developing countries. The generic fixed-dose combination of nevirapine, stavudine, and lamivudine (Cipla, Mumbai Central, Mumbai, India) is one of the most frequently prescribed treatments in African countries. In Angola, a south west African country with more than 15 million inhabitants, the prevalence of HIV infection is increasing rapidly, with up to 8% of town-dwelling pregnant women infected (UNAIDS 2002). HIV-1 predominates, and HIV-1 groups M, N, and O and many subtypes and circulating recombinant forms co-circulate (Vergne at al 2003). A national antiretroviral access programme has started, most usually based on the generic combination of nevirapine, stavudine, and lamivudine owing to its low price. However the sad fact remains that many AIDS-infected patients in Angola go without any anti-viral treatment. Use of these combination therapies has dramatically decreased morbidity and mortality rates in HIV-1 infected individuals (Hogg et al. 1997; Cameron et al. 1998; Palella et al. 1998). However, such intensive combination therapies have various drawbacks, such as drug side-effects, the complexity of the therapeutic regime, and the appearance of resistant HIV strains (Carr et al. 1998; Colgrove et al. 1998; Sarmati et al. 2002). So far, it appears that these combination anti- retroviral drug regimens must remain in use until a radical curative treatment and
HIV-1 vaccine are established. Therefore, therapies based on new principles
other than drug treatment would appear to be highly desirable.
Electrical stimulation effects on living cells have been extensively studied since
the 1970s (Zimmerman et al. 1974). It has been reported that use of high d.c.
voltage pulse application not only induces changes in cellular membrane
structure and permeability, but also results in its breakdown (Berg et al. 1984;
Powell et al. 1986). Moreover, cells suspended in solution were found to be
easily fused by pulses of high d.c. voltage (Zimmerman 1982). On the other
hand, pulses of low d.c. voltage are known to regulate cellular proliferation and
protein production together with induction of differentiation of various cell types
(Kojima et al. 1992; Mie et al. 1996; Aizawa et al. 1999).
Furthermore, the effects of electrical stimulation on HeLa cells chronically
infected with HIV-1LAI and on uninfected P6 HeLa cells (Tominaga et al. 2003)
has been reported, and the possible causes for the significant damage that
occurs to infected P6 HeLa/HIV-1LAI cells compared with uninfected control cells
has been considered.
In this study we tested the effectiveness of an electrotherapy apparatus (The Bio-
Lyfe machine), that delivered an oscillating electrical signal via hand-held probes,
on the immunological parameters and viral load in HIV infected patients.
Methods

All participants in the trial were periodically monitored with respect to their clinical
features; any side effects were noted. Blood was withdrawn and placed into
tubes with ethylenediaminetetraacetic acid (EDTA) as anticoagulant; the plasma
was used in tests and some was also stored at -70 C. Laboratory tests,
including routine blood tests, and CD4 and CD8 counts, were performed at the
Military Hospital. The HIV viral load was determined in the HIV Laboratory,
Laboratory Services Branch, of the Military Hospital which used the sensitivity
cut-off value of 10 000 HIV copies of RNA per millilitre) (Chiron Diagnostics
1997). All data and clinical records were kept confidential.
In this study, 37 patients with HIV infection were treated with The Bio-Lyfe
machine. The treatment period was at least 2 months. Patients were exposed to
hand held electrode probes daily for 20 minutes at the highest tolerable current.
There were no obvious side effects reported throughout the two months.
Compliance was difficult to maintain as it involved patients coming to a medical
centre daily. Seven patients were discontinued from the trial due to poor
compliance. Thirty HIV infected patients who were not participating in any anti-
HIV treatment were randomly chosen from the hospital database and matched
with trial patients who were observed for two months.

The CD4 and CD8 counts and HIV viral loads of patients were measured before
the trial, at 1 month and at 2 months of treatment with the Bio-Lyfe machine
(Tables 1 and 2). These results were compared to test results for the untreated
patients. All patients gave their written informed consent.


Raw Data
Table 1 Treated Patients:

Table 2 Untreated patients:

Statistics

The purpose of the study was to study the use of electrotherapy on HIV-1 patients. There
were 67 patients enrolled; 53 of these completed a 2-month period of involvement in the
study. Of these, 26 received a daily oscillating electrical signal treatment over a period of
at least 2 months, while 27 (the controls) did not.
The patients to be treated were hand-selected by the researcher from the hospital
database, with the intention of selecting a representative range of values for the outcomes
of interest, CD4 and CD8 lymphocyte count and viral load. The controls were randomly
selected from 80 of the remaining patients on the database who were deemed to be
similar in age and gender to those selected to be treated, though they were not
individually matched. Patients were excluded from the study if they were already
receiving an alternative HIV treatment. All 53 patients were monitored over a 2 month
period, and measurements of CD4 count, CD8 count and viral load were made at 0, 1 and
2 months.
In this report, we present analyses to address the question:
• Is there a difference in the CD4 count, CD8 count and viral load between patients who received electrotherapy and those who did not? Method

The data were provided in two files, one for treated and one for untreated patients. The
following information was provided for each patient: gender, age and measurements for
the three outcomes at each of three time points.
Since the selection of patients for treatment was not a standard random selection, there
could have been bias in the selection process. Although it is difficult to dispute this
point, initial measurements for the treated and untreated groups were compared, which
would be expected to be similar if there was no selection bias.
For each outcome, the main variable of interest was the change between the first
measurement and the 2-month measurement, as a percentage of the first measurement.
The percentage change was of interest because there was a large amount of variability in
the measurements between patients. The two groups were compared separately for the
three variables of interest. As the data did not appear to be normally distributed, non-
parametric tests were used for these comparisons. The particular test used in each case
was the Mood test, which makes no assumptions about the distributions of the two
outcomes. Other, less conservative tests could have been used and did, in fact, indicate
more significant differences between the groups, but these relied upon assumptions which
did not appear to be valid when visual representations of the data were considered.
Comparing initial values for the two groups
The gender representation in the groups was almost identical (2 females in each) and the mean ages were not statistically significantly different between the two groups (29 years for the untreated compared with 28 years for the treated group). Dotplots of the initial values for each of the three outcomes are given below, along with a table of descriptive statistics for these outcomes. None of these differed significantly between the groups, although there was a greater spread of values for viral load in the treated group, probably due to the way that these patients were selected. This difference may have implications for the final results. Dotplots of initial CD4 and CD8 counts and viral load
Standard
Outcome Group
deviation Minimum Median Maximum
Comparing the treatment effect over time
Below are the results for the three outcomes. In each case, the same information has been provided: a dot plot comparing the percentage changes for the two groups, summary information of the percentage change for the two groups and the results of the Mood test comparing the median percentage change for the two groups. For each test, a P-value has been given, which is the probability of observing the difference in medians that we have or a more extreme difference, assuming there was no underlying difference. Obviously a small P-value is evidence of a difference between the groups and a P-value is usually considered to be statistically significant if it is below 0.05. CD4 lymphocyte count
Dotplot of percentage change in CD4
e
Tr
Yes-48
% change in CD4
Summary statistics for percentage change
Standard
deviation Minimum
Median Maximum
Treated 26 5.0 25.9 –29.1 –4.3 70.0

Results of the Mood test
Difference between medians (untreated minus treated) = –11.2
95% confidence interval for difference between medians: (-22.6, 0.4)
CD8 lymphocyte count
Dotplot of percentage change in CD8
% change in CD8
Summary statistics for percentage change
Standard
deviation Minimum
Median Maximum
Treated 26 18.3 42.3 –26.3 4.0 147.3

Results of the Mood test
Difference between medians (untreated minus treated) = –17.4
95% confidence interval for difference between medians: (-41.1, -2.1)
HIV viral load
Dotplot of percentage change in viral load
% change in viral load
Summary statistics for percentage change
Standard
deviation Minimum
Median Maximum
Treated 26 –5.9 69.1 –91.9 –20.5 248.1
Results of the Mood test
Difference between medians (untreated minus treated) = 57.9
95% confidence interval for difference between medians: (18, 109)
Conclusion
Our tests found that there was a statistically significant difference between the two
groups for the outcomes of CD8 count and viral load. The treated group had:
• a small positive median percentage change in CD8 lymphocyte count, compared to a large negative median percentage change for the untreated group; • and a large negative median percentage change in HIV viral load, compared to a large positive median percentage change for the untreated group. In these results, there was a consistently better outcome in the treated group, although the difference in median CD4 count was not significant. There are important reasons why it is difficult to infer from these results that the treatment generally causes a beneficial effect. As the patients selected to receive the treatment were hand-chosen in order to provide a good range of values (while the controls were a simple random sample), differences between the groups may be the result of more than just the electrotherapy. For example, viral load was generally larger in the treatment group and it could be argued that a larger initial value generally results in a larger decrease in viral load over time. Also, even though the patients were selected from the same population, the specific nature of this population does have implications for any inference that can be made. It is not possible to tell how patients in a military hospital may differ from the general population, but it is highly unlikely that this population is representative of the wider HIV-1 population of Angola. Graph of HIV Viral Load Changes Over 2 Months for Individual
Patients in the Treated Group

HIV Africa Treated Patients
Patient number
Graph of Change in HIV Viral Load Over 2 Months for Individual
Patients in the Untreated Group

HIV Controls Viral Load
Patients
CD4 + CD8 Graph – Summary Results - % Change over 2 months
HIV Africa 2005
Viral Load Changes Graph – Summary Results

HIV Electrotherapy Study Africa 2005
% change of viral load over 2
Patient Groups
Discussion
There is a positive effect of treatment with The Bio-Lyfe electrotherapy machine on CD8
cell count followed by and a negative significant effect on the viral load viral load a
positive non significant effect on CD4 count in many of the HIV-infected patients. Large
individual variations in response were also observed. We have previously observed
positive effects in CD8 cell counts in various cancer patients and in three HIV patients
(unpublished data).
The multi wave oscillating device provides an electrical signal to two hand held
electrodes that boost CD8 immune system cell numbers.
By boosting these CD8 cells the viral load is reduced, and the treated patients experience
an improvement in the quality of life and an extension of life.
Research at Duke University has shown that high levels of CD8 cells have a positive
effect on prognosis in AIDS-infected patients. The proposed mechanism of action is that
in asymptomatic patients, CD8 cells can identify infected CD4 cells, latch onto them, and
release compounds that cause the infected cell to burst, killing it.
CD8 cells also possess a non-cytolytic weapon as well, and this stops HIV replication.
Specifically Tomaras and Greenberg’s experiments (2000) at Duke University show that
CD8 cells affect the virus after it has already entered the CD4 cell, which is different to
the way beta-chemokines work. The CD8 cells stopped HIV from hijacking the CD4
cell’s genetic machinery to reproduce itself.
Discussion Of Other Relevant Studies

Immunologic characteristics have previously been studied in 103 patients with multiple
myeloma, acute leukemia, chronic lymphocytic leukemia and non-Hodgkin's disease
following in vitro exposure of blood to a low-intensity static field (SF) and alternating
field (AF) or pulsating magnetic field (PF) (Bessmel'tsev 2001). In this study of multiple
myeloma, a 30 minute exposure had a positive effect on expression of tumor cells and T-
cell markers and stimulated the regulatory function of T-lymphocytes. With SF-AF and
PF application alternating, the expression of both +CD3 and +CD4 and the +CD3/+CD4
ratio increased suggesting the lowering of immunological deficiency. In acute leukemia,
a combined application of the magnetic fields had an effect on the helper activity of the
T-lymphocyte sub-population. The phagocytic activity of leukocytes increased
significantly while their digestive ability rose to a moderate degree.
The effect of leukocyte subsets, total leukocyte isolates or full blood samples subjected to
medium-strength square-wave electric impulses (100 V/cm field force, 5 ms duration) has
also been reported (Filipic 2000). On the surface of the leukocytes, the expressions of
several markers (CD3, CD4, CD8, CD11a, CD11b and ICAM-1) were determined in
order to study the influence of pulsed ionic currents on different aspects of the cellular immune response. Large individual differences were also observed in this study among randomly chosen healthy donors, both in the initial expression rate and in the response patterns of different antigens. As a general conclusion, it can be stated that electric impulses with the above parameters activate the state of immune response alertness of human leukocytes. Elevation in the activities of several enzymes, such as lactate dehydrogenase and superoxide dismutase in the serum in response to electric impulses suggested an antiviral and immune activated condition. It was concluded that ex vivo blood treatment with medium-strength electric impulses seems to be a promising adjuvant course for the establishment of acute immune potentiation and an antiviral state in patients undergoing dialysis treatment. The effects of uncontrollable and controllable electric shocks on the immune system in rats (the proportion of CD4+, CD8+ or CD25+ T lymphocytes to total lymphocytes was measured in the peripheral blood, spleen, and thymus) have also been observed (Nakata 1996). The rats were given either controllable shocks, identical uncontrollable shocks, or no shocks, and then small shocks 24 h later (reinstating shocks). The proportion of CD4+ T lymphocytes relative to total lymphocytes in both the peripheral blood and spleen of uncontrollable rats (URs) was significantly smaller than was found in no-shock rats (NRs). Similarly, the proportion of CD4+ T lymphocytes in the thymus of controllable rats (CRs) was significantly smaller than in NRs. In contrast, the proportion of CD8+ T lymphocytes in the thymus of URs was significantly larger than in NRs and CRs. The CD4+ to CD8+ T lymphocyte ratios (CD4+/CD8+ ratios) in the peripheral blood, spleen and thymus of URs were significantly smaller than in NRs; also, the ratios in the peripheral blood and spleen of URs were significantly smaller than in CRs. The white blood cell (WBC) count of URs was significantly smaller than those of NRs and CRs, and the WBC count of CRs was significantly smaller than those of NRs and CRs, and the WBC count of CRs was significantly smaller than that of NRs. These results suggest that decreases in CD4+ T lymphocytes (and/or an increase of CD8+ T lymphocytes) in the peripheral blood, spleen, and thymus are caused by uncontrollable stress followed by a reinstating stress condition, leading to the decrease of WBC in the peripheral blood and decreases in the CD4+/CD8+ ratios in these tissues. Electrical stimulation can also affect neuropathies in nerves and circulating neurotransmitter levels (Herzberg 1995). This indirectly can affect the immune system (Lechin 2002). To study the possible mechanism by which peripheral nerves mediate immune responses in target tissues, electrical stimulation of the sciatic nerve was combined with subcutaneous microdialysis of the hind paw (Herzberg 1995). Following unilateral stimulation of the sciatic nerve, an ipsilateral rise in substance P and a bilateral rise in VIP levels were observed in dialysate samples from experimental vs control animals. Electrical stimulation of the sciatic nerve induced a marked hyperemia and swelling of the ipsilateral paw. Quantitative immunocytochemical analysis of paraffin-embedded sections of the hind foot pads demonstrated T lymphocyte migration ipsilateral to the stimulated nerve. These findings suggest that peripheral nerves can directly modulate local immune and inflammatory responses. It has been shown in previous studies that cell poration (i.e. reversible permeabilization of cell membrane) and cell fusion can be induced by applying a pulse (or pulses) of high-intensity DC (direct current) electric field (Chang 1989). The same group also suggested that such electro-poration or electro-fusion can also be accomplished by using an oscillating electric field. The DC field relies solely on the dielectric breakdown of the cell membrane to induce cell fusion. The oscillating field, on the other hand, can produce not only a dielectric breakdown, but also a sonicating motion in the membrane that could result in a structural fatigue. Thus, a combination of a DC field and an oscillating field is expected to enhance the efficiency of cell poration and cell fusion. Pulses of high-intensity, DC-shifted RF (radio frequency) electric field were used to induce a highly efficient cell poration and cell fusion on human red blood cells and on a fibroblast cell line. Another study demonstrated a safe and effective way to introduce exogenous genes into cells, a new method of electroporation which uses a radio-frequency (RF) electric field to permeabilize the cell membrane has been uncovered (Chang 1991). This RF method has several advantages over the conventional electroporation method which uses a direct current (DC) field. It was shown that the RF electroporation method can be used to introduce marker genes into a wide variety of cell lines and was able to increase substantially the efficiency of gene transfection. Interestingly, the transfection efficiency was shown to be affected by a number of factors, including cell type, field strength, pulse protocol and medium buffer. Because of its wide range of applications, high transfection efficiency and lack of harmful side-effect, the RF electroporation method would be particularly useful for introducing genes into human cells for gene therapy. Morphological changes of HIV infected cells following application of low electrical potential were induced due to a decrease in the plasma membrane fluidity and deformation of cytoskeletal structure including F-actin (Aizawa et al. 1999; Yaoita et al. 1989). It has also been suggested (Kumagia 2004) that the swelling and breakdown of cell membrane due to application of electrical potential might have occurred due to an increase in the adhesion area between the positively charged ITO electrode and the negatively charged cells after electrical stimulation. Several other factors possibly contribute to the higher cellular damage due to electrical stimulation in P6 HeLa/HIV-1LAI cells than in P6 HeLa cells. It was considered that the most important factor to be the large surface area of chronically HIV-1 infected cells due to the budding of HIV-1 particles at the plasma membrane, which is not observed in uninfected cells (Barré-Sinoussi et al. 1983; Chrystie and Almeida 1988). In other words, budding of HIV-1 would increase the negative charge of infected cells, and hence lead to increased reception of the effect of electrical stimulation. Another factor would be that some oxidized species might have developed due to the electrochemical reaction at the ITO electrode surface in the supernatant of P6 HeLa/HIV-1LAI cells. Collectively, it has been shown (Kumagai 2004) that the difference in sensitivity to electrical stimulation between uninfected cells and cells chronically infected with HIV-1 could be useful not only for the detailed elucidation of HIV control mechanisms but also for the development of new therapies.

Summary - In Vivo Pulsed High Potential
Electrotherapy

Pulsed high potential electrotherapy technology appears to offer a novel approach to
treating diseases related to immunosuppression, such as infection with HIV.
The technique involves the use of brief high potential spikes which disrupt the skin’s
dielectric barrier and allow specific electrical potentials to penetrate into the body’s
tissues. These potentials carry specific waveforms and frequencies which can have
positive effects on the healthy function of a range of tissues in the body. In addition these
high electrical potentials can disrupt the weakened membranes of infected cells, causing
cell death. These potential also appear to have a specific anti-microbial effect. It is
thought that electromechanical and electrostatic stresses are induced by these high
voltage pulses at multiple audio and radio frequencies.
The electromechanical stresses, cell electroporation, and ion exchange processes induced
by high voltage DC pulses are well documented in vitro. These processes may act to
both destroy infected cells and to stimulate the immune system. (Bryant 1987, Chang
1989, Dao-Sheng 1990, Dimitrov 1990)
Inactivation and destruction of bacteria by electric treatment methods is well established
in vitro. In addition pulsed high voltage electrotherapy at multiple frequencies appears to
inactivate pathogenic micro-organisms in vivo. (Allen 1966, Hamilton 1967, Gillibrand
1967)
In addition high potential electrotherapy is showing promise in treating inflammatory
conditions (Johnson 2004), and in treating cancer – both as a direct cytotoxic agent
(Fedorowski 2004, Hernandez-Bule 2004, Kirson 2004, Pinero 1997) and also by means
of enhancing the efficacy of chemotherapy and radiotherapy through electroporation
effects on the membranes of cancer cells (Ito 2001, Engstrom 2001, Gray 2000, Li 2003,
Rabussay 2002, Rols 2000, Sun 2003).

Conclusion

There are multiple studies in the literature suggesting that electrical stimulation can
induce positive immunological changes both in vivo and in vitro, and negatively effect
HIV-infected cells with no effect on non-infected cells.
The Bio-Lyfe electrotherapy apparatus seems to have an obvious positive effect on HIV-
infected individuals in this pilot study.
However it’s mechanism of action for inducing these changes is currently unclear.
Further more detailed and larger trials on the efficacy and safety of The Bio-Lyfe
machine are warranted, as are studies into the possible mechanism of action of The Bio-
Lyfe machine.
It is of note that The Bio-Lyfe machine appears to be a cheap, safe and easy to use
treatment system. It may even be appropriate for home use.
The Bio-Lyfe machine looks to be a real possibility to add to the very limited arsenal of
weapons to fight the current global HIV epidemic.
Further studies are therefore warranted.
Acknowledgements:
Statistics were prepared by Sandy Clarke BSc(Hons) GStat, Statistical Consulting Centre,
University of Melbourne.
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Bio-Lyfe Machine
Index
Investigation of the effect of a multi wave oscillator in the treatment of adult HIV/AIDS patients: . 1 Abstract . 1 Introduction. 1 Methods. 3 Results. 5 Raw Data. 5 Table 1 Treated Patients: . 5 Table 2 Untreated patients: . 6 Comparing initial values for the two groups. 8 CD4 lymphocyte count . 9 CD8 lymphocyte count . 10 Graph of HIV Viral Load Changes Over 2 Months for Individual Patients in the Treated Group . 13 Graph of Change in HIV Viral Load Over 2 Months for Individual Patients in the Untreated Group. 14 CD4 + CD8 Graph – Summary Results - % Change over 2 months . 15 Viral Load Changes Graph – Summary Results . 16 Discussion . 17 Discussion Of Other Relevant Studies. 17 Summary - In Vivo Pulsed High Potential Electrotherapy. 20 Conclusion . 20 References. 21 Index . 26

Source: http://ftp.smile.org.au/Angola%20Electrotherapy%20Study%20-%209%20March%202006.pdf

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