Referees comments about the PhD thesis of Michaela Pekarov titled Role of

arginine and its metabolism in physiology of macrophages.

The dissertation comprises the summary of the most important results of the
applicant. The summary was followed by a detailed literature list in the first part.
The next part of the dissertation consists of copies of full size publications the
nominee has already published. Two of these papers serve as the basis for the
present dissertation, a methodological description and application to quantify the
concentration of nitric oxide (NO), and the role of arginine in endotoxine (LPS)
induced activation of macrophages. The second part of the publications consists
of nine other papers already published in internationally accepted journals which
are also connected thematically to the main topic, i.e. to macrophage and
endothelium derived ROS and RNS production and the regulatory function of
these molecules in immune functions.
The structure of the dissertation fits the requirements of the university. I was not
able to find formal mistakes to blame.
The text is written in English language. I have nothing to comment on the
already published papers because they have already been reviewed by authentic
persons. However, there are a couple of misprints and also, in some cases,
incorrect usage of the English language. At the same time I have to admit that
that these mistakes are not frequent and they never interfere with understanding
the text. I also have to admit that not being a native English speaker; I do not
think I have the right to heavily criticize other colleagues usage of the language.
Beside that the text is well composed, it is easy to understand, facts are clearly
explained and also they are well documented.
Scientific evaluation of the work. The role of arginine in the regulation of
macrophage functions is very important. The role of the immune system in
different homeostatic functions has become more and more important. Although
we speak about a system still the execution of different functions are mostly
attributed to particular representative cells of it. And also these cells are not
organized in solid organs. One of the mostly studied cell line is the MO/M
system, which is responsible for innate immunity, the first defense system
against invaders like micro-organisms or any other substances that provoke a
defense response against molecules recognized as foreign substances. In this
aspect the study of any of any cell functions of the system is very important. The
immune system is somewhat different from other organ systems because it is not
organized in solid organs, but it comprises of different cell lines which have
individual functions and also in some context their functions are individually
regulated. This so called individualism gives us good opportunity to study the
function of these cells and cell lines. Several such lines exist that resemble to the
originals immune cells. On this basis the choice of RAW 264.7 was very good.
Different immune cells require different nutrients among them carbohydrates
and their metabolites but other substances like fatty acids and amino acids
among them glutamate or arginine and probably others are also essential. In this
context the choice of the amino acid, L-arginine is interesting. Arginine seems to
be essential in regulating Mo/M functions for some reasons: 1. It is the
substrate for the enzyme, nitric oxide synthese (NOS) to produce NO an
important regulator of several cellular functions consisting immune and other
cells of the body. 2. L-arginine is also an important substrate for cellular
metabolism as it has been reviewed by the author. 3. The latest concept which
was also mentioned by the nominee amino acids and particularly L-arginine can
become not only a substrate of different cellular metabolic processes, but it can
also serve as a signaling molecule. 4. Arginine supply under physiological
conditions is supposed to be adequate; however, if increased needs are present
and/or the supply is limited, like in increased iNOS activity or in limited oxygen
supply (hypoperfusion), respectively, its availability might be also limited. In
these situations, however, intracellular priorities are not known yet. This
important question was also raised by the author, but it has been left unanswered
because of lack of sufficient knowledge.
In summary the choice of topic and questions to be answered are very important
and can increase our knowledge about the regulation of immune functions.
Most important scientific results.
1. The research group has developed a special micro method to measure NO
production and NO concentrations in biological samples. They have developed
the modified porphirinic sensor by the use of which NO production was
monitored electrochemically using a potentiostat.
2. Increase in extra-cellular arginine concentrations enhance the activation of all
MAPK pathways in RAW 264.7 macrophages. This activating role of L-arginine
is dose dependent and it is further increased if RAW 264.7 cells are stimulated
by LPS.
3. Extra-cellular arginine regulates further down-stream steps of macrophage
activation. Extra-cellular arginine is needed for the full activation of LPS
induced NF-B activity in macrophages.
4. LPS and arginine stimulated macrophages (RAW 264.7 cells) produced
increased amounts of NO due to an increased iNOS protein production and its
activation.
5. Down-regulation of iNOS protein expression is associated with reduction of
O2. production in LPS stimulated macrophages and this process was potentiated
by increased extra-cellular arginine concentrations.
Evaluation of the methods:
As it has already been mentioned the immune system is composed of individual
cell lines which certainly have important interactions with each other. However,
one of the most important and to date the best methods are based on the
examinations of individual cell lines in cell cultures. Among the possibilities the
RAW 264.7 cell line is an excellent tool to study the important biological
reactions of immune cells.
All other methods, chemical physico-chemical determinations, immunological
methods, methods of molecular and cellular biology have been properly applied
and correctly executed. The methodological armory was proper to get answers
for the scientific questions. Detection of NO concentrations in micro-liter
samples has to be particularly appreciated, which is also a major achievement of
the present work.
Comments and questions: Most of the works presented in the attachments have
already been published in international journal, in other words they have been
revised and corrected. Therefore it is difficult to find any short comings.
1. What was the reproducibility of NO concentration determinations?
2. How did you determine absolute (milli molar) values of NO concentrations?
3. Peak signal following addition of NO in the measuring chamber was reached
in 155 sec. If you had SEM values, the scatter is too high. What is the
explanation to that?
4. Did you test the sensitivity of the NO electrode in the long run (hours) by
simply adding the same volume of NO saturated test solutions.
5. You had NO record for several hours. How could you make sure that there
was no loss of sensitivity of the electrodes?
6. Have you tried to differentiate between NO amounts produced by eNOS and
iNOS, respectively? Selective iNOS/eNOS blockers could make it possible.
7. In the first manuscript you gave a detailed description of all methods with the
exception of the determination of intra-cellular arginine concentrations. It would
be very important because this parameter is very informative.
8. How do extra-cellular (physiological or pathological) L-arginine
concentrations relate to cell culture studies you used?
9. Did you control or measure arginine concentration changes throughout the
studies, particularly in experiment lasting for several hours?
10. Intra-cellular arginine is a substrate for at least two enzymes (iNOS and
arginase). Do you know how these two ways can become preferential? (How
high are the Km values?)
11. You gave an excellent flow chart on page 16. This is the summary of how
LPS activates intracellular processes in macrophages. Another suggested chart, a
summary of arginine metabolism and ways of its actions would have helped
understand this problematic.
12. iNOS can produce both NO and O2. depending on arginine supply. Is there
any change in the molecular structure of the enzyme? (Analogy Xanthine-
oxidase/dehydrogenase conversion in hypoxia.) If there is any change in the
molecule, is this change reversible?
13. The concept of amino acid receptors is interesting, although it is a
speculation. Is it possible that the same structure is responsible for transport and
trans-membrane signaling? I do not think so. If there are different molecules (or
structures) present in the membrane which mechanisms could differentiate
between the two possible ways?
14. Is it only iNOS which can produce both NO and superoxyde anion or has
eNOS the same capacity?
As a summary of my comments I can state that Michaela Pekarov has carried
out excellent research in different laboratories, she is already a well trained
researcher capable to carry out scientific activity alone. She has studied
important scientific questions, to answer these she has used proper methods and
also developed a sensitive method to measure NO concentrations. Results of her
research have been published in acknowledged international journals. On the
basis of all her achievements I strongly support her application for the PhD
procedure and also recommend the donation of the Dr. title.
Budapest, 17.- 07. 2010.
Sincerely,
Jnos Hamar MD. PhD.