Transcript (1526 bp) synthesized on template bacterial DNA of AIDS patients

The role of human
microbiome in AIDS
process
Zajac V., Ciernikova S., Wachsmannova L.,
Adamcikova Z., Stevurkova V., Krcmery V.
Cancer Research Institute, SAS, Bratislava, Slovakia
Saint Elizabeth University of Health, Bratislava, Slovakia
Introduction
Despite great success in the diagnostics and therapy of AIDS, there
are many unanswered questions. Without giving the answers to these
questions more successful treatment of patients can not be expected.
The strong argument for this prediction is a fact that it is not possible
to stop the worldwide pervation of AIDS, especially in Africa and Asia.
The data leading to the conclusion that HIV alone is the etiologic agent
responsible for AIDS is generally accepted. According to this claim,
virus was transferred to humans from monkeys in Africa through
random contacts 35 to 50 years ago, which was not sufficiently
confirmed.
Plasma HIV RNA is dramatically reduced in patients treated with
HAART, but residual viral replication is detected after suppression of
plasma viremia. It has also been proven that various forms of HIV
reservoirs persist in practically all patients receiving this therapy.
Reservoirs were detected in macrophages and other cells of the blood
system.
• The range of viral reservoirs in the human body is probably very much
wider, as is claimed in recent studies. The source of persistent HIV in
infected persons remains however unclear.
GIT, bacteria and AIDS
• There is increase of evidence, pointing out that in the GIT and
other mucosal tissue, and not in the blood, is the main place of
HIV infection and CD4+T cells loss. These findings go along with
new studies on the role of bacterial translocation in the gut as
central driver of AIDS pathogenesis.
• This interest in investigating bacteria and mycoplasma in this disease
was also supported by Montagnier’s finding, confirmed by Shyh-Ching
Lo, that mycoplasma is a very important “co-factor” which accelerates
the progression of HIV infection in AIDS patients
• Bacteria and yeasts represent second kingdom in our body. The
fact that the balance between two kingdom is the basis of our
existence is still underestimate.
• This balance, established for many centuries, was during the last
decades seriously disrupped by application of ATBs, pharmaceuticals,
drugs and changes in life style (anal sex).
• There are new studies on the role of bacteria to antibiotics
in diseases associated with AIDS. It demonstrates clearly
that bacteria can induce in the gut and the vagina
transcription of silenced genes, including HIV-1 provirus.
• The HIV-1 has been also detected in the bowel crypt cells
and lamina propria. Since these cells are in close nearness
to intestinal bacteria, the idea that bacteria can also be
involved in the pathogenesis of AIDS is justified. Bacteria
are a strong candidate for a viral reservoir in the human
body.
• These findings are challenging for us to fight against AIDS
in a more complex manner.
• It is highly desirable to overcome all dogmas and taboos
surrounding the disease. In advance, it is necessary to
deliberate another potential factors, not only HIV, which
may take a part in this disease.
Our approach
• We study the role of bacteria and yeasts in bovine leukemia and
concequently in AIDS process for more than 25 years.
• This approach is based on detection of BLV-like sequences in bacteria
of BLV positive animals.
• Gradually been identified HIV-like sequences in laboratory of prof. F.
Wong Staal, UCSD and later HIV-like proteins in bacteria and yeast in a
cohort of 80 HIV positive patients from Slovakia, USA, Kenya and
Cambodia.
Methods used for detection of HIV-like sequences and HIV-like
proteins:
- Colony and dot-blot DNA hybridization
- PCR
- sequencing
- Western blotting
- GPA (gentamicin protection assay)
Dot blot hybridization of lymphocyte's and bacterial DNA
of AIDS patients. Probe: PCR products 38;39 and 68;69
synthesized on the template of patient's lymphocyte DNA
PCR products (1526 bp) synthesized on template
bacterial DNA of AIDS patients
1 2
3
4
5
6
7
8 9 10 11 12 13 14
15
Lines 1-12: M1, P3, P6, P9, P15, Mok1, M11, M12,
M15, M21, Mok22. Negative controls in lines 13.
PCR without DNA (line 14). Control pBH10 in
line15. Used primers: O1,O2 (HIV-1 env gene).
Sequences of 348 bp PCR product syntesised on the template of bacterial
DNA isolated from AIDS pacient P3, limited by primers O1,O2 (5837-7297) of
env gene HIV-1. Identity for 95% with HIV-1 isolate HXB2.
Western blotting of proteins isolated from bacteria and yeasts of the respiratory tract
(nose, pharyngeal swabs) of Cambodian (Km) and Kenyan (Ke) HIV positive children.
Used monoclonal antibodies against HIV-1 gp120 (1:750). lines 1-6 tested samples:
14Km, 17’Ke, 21Ke, 32’Km, 3’Km, 31Km, 14’Ke line 7: serum of AIDS patient
diluted 1:100; line 8: control bacteria; line 9: negative control bacteria Muta 104-0.
Internalization of HL-60 cells by bacteria of AIDS patients and
patients with colorectal tumors (GPA)
Patient/clone
P15/7
P1/4
P3/3
Mok12/5
Mok 1/6
K1-1
Number of bacterial colonies
2264
1340
1680
lysis of HL-60 cells
181
lysis of HL-60 cells
TuSG
71
883 S
104
negat. control
<5
-----------------------------------------------------------------------------------------Number of HL-60 cells 2x106/ml
Number of bact. cells 0,8x108
Internalization of normal human lymphocytes by bacteria of
AIDS patients (GPA)
Patient/clone
Number of bacterial colonies
P15/7
1121
P1/4
complete lyses of human lymphocytes
P3/3
complete lyses of human lymphocytes
Mok12/5
complete lysis of human lymphocytes
Mok 1/6
complete lysis of human lymphocytes
K1-1
complete lysis of human lymphocytes
Mok 22/5
423
725/5
1140
-----------------------------------------------------------------------------------------Number of lymphocytes 2x106/ml
Number of bact. cells 0,8x108
Probiotic treatment of 20 patients
(Nissle 1917)
Viral load in time of probiotics treatment start:
808 600
Viral load in term of the end of probiotic treatment: 452 190
Decrease of viral load after probiotic treatment:
Viral load of 11 patients from 20 decreased (60%)
356 410
(44%)
The origin of HIV
According to our and other results there is strong
objection again generally accepted dogma that HIV was
transmitted to humans from apes in Africa about 35-50 or
according to last assertion about 100 years ago en route
of accidental contacts. That is a good news for Africa.
There is not evidence about transmission of retroviruses
between two different species in nature. Should be HIV
only one exception in nature? Are there adequate proofs
for this claim?
The problem is that the acceptance of this dubious
argument has a major influence on – research, diagnosis
and therapy of AIDS.
How is it possible that this unconvincing statement was
accepted by most experts???
Our hypothesis
HIV is an integral part of humans since the beginning of our existance.
We inherited it from our ancestors. Bacteria and yeasts are most likely
natural host of HIV sequences.
• But, there is serious objection – if HIV was in our body from our
beginning, why did it’s emerge just about 25-35 years ago? To answer
this question we should go back into the ancient history of humankind.
In the past, major epidemics frequently occurred when there were new
patterns of transport between separately populated areas and new
pattern of settlement. This tremendous longtime „sanitary“ process
take place mainly in Europe, consequently in USA, GB colonies,
partially in Asia and north Africa resulted in establishment of the
balance between these two kingdoms in the human population.
• This balance was interrupted – intestinal dysbiosis - in the middle of the 20.
century due to acceptation of ATBs, drugs (including recreational), medicines
and changes in life style (anal sex). Propagated HIV bearing pathogenic
microbes in the majority of antibiotics resistance, with the ability to pentrate
into the body.
When bacteria carrying the HIV sequences with high affinity to lymphocytes
penetrated into the blood, infected or lyse them, consequently the immune
system has collapsed. The result of this process is ..... AIDS
This hypothesis is based on:
- respecting the central role of HIV genetic information in the
process of AIDS
- claims that bacteria and yeasts are most likely natural host
of HIV genetic information
- recommendation that in AIDS it is necessary to deliberate
another potential factors, not only HIV, which may take a
part in this disease and may be different in various groups
- assumption that transmission of HIV from apes to humans
during last decades in Africa as a consequence of their
accidental contacts - is not a cause of AIDS. Bubonic
plaque epidemic in 1346 very probably induced high
frequency CCR5 mutation in the European population.
Confirmation of our hypothesis may opens new opportunities
for research and treatment of AIDS. Only through open
discussion and respect for different views may lead to the
elimination of this disease.
and now, we are able to
answer on many until now
unanswered questions
Presented hypothesis answer to many until
now unanswered questions:
- origin of HIV
- large scale HIV positivity in Africa
- connection of AIDS with TBC in Africa
- absence of „gold standard“ in Africa
- specificity of AIDS in USA
- the presence of HIV reservoirs after antiretroviral therapy
- atypical course of disease in comparision with other
retroviral infections
- the rarity of complete viral particles detection in AIDS
patients, but detection of HIV sequences and the HIV-like
proteins herein
- toxicity of some anti-HIV drugs – AZT and other
Dot-blot hybridization of bacterial DNA (0.25 μg) from Cambodian HIV positive children.
The hybridization probe was mixture of PCR products that represented gag, pol
and env HIV-1 genes synthesized on the template of plasmid pHB10. Samples of 39
patients were applied in lines from A to G. The samples of 8 healthy persons are
in lines H and I. In the last line J in position 6 is DNA of tested child with shining
clinically expression of disease and in positions 2, 3 are mixtures of aforementioned PCR