parasites

I am blogging again. I am going to investigate the types of intracellular parasites infecting many poeple with chronic illnesses or irritants.
I will examine stool specimens if peope bring them to me. I can record findings with microscopic equipment.

Treating toxo and malaria with herbicides?

Could herbicide fluridone be used as therapeutic treatment of certain apicomplexans infecting humans?

My new office

These are pics of my new microscopy mini lab. I am open for biz!

My Apologies

I am in the process of setting up an office in the research district of Toronto. I will be open for business very soon, and will except appointments.
Will be networking with other researchers, so opportunities will exist to find answers, at least, to some of the pressing questions about acquired chronic physical symptoms in healthy people.
My focus will be on immunology, and pathological consequences of self verses non-self antigens.

Apicomplexia complexity

I suspect there are cross-gene species of apicomplexans that may be worth looking at, in certain clinical situations.
I think there needs to be expansion of available primers for spp. such as plasmodium.

Staining for fecal oocysts averaging at 3-4 micrometers should be emphasized in stool tests of patients with autoimmune symptoms.

New apicomplexan\other parasite?

I have some interesting information to reveal "soon" regarding some intensive DNA sequencing of my parasite which was submitted weeks ago. Information from my own documentation of this microbe is in earlier postings.

This information will interest those studying apicomplexia, their genetic diversity and ability for quroum sensing.

My Microscopic research studio is now open for business in Toronto.

Continuation of culture evaluation

A typical cluster.






This one is weird it has interesting internal morphology.
I have seen others without pigmentation, and many moving entities inside the membrane. If I see more like those, I will post video.






Typical cell. I can't even tell if it grows from the "rosegerms" shown in previous post's micrographs.




These micrographs show developing structures, that I admit, were unexpected, and completely unexplainable, at least so far.
I have made some adjustments to the growth medium, by infusing carbon dioxide into the broth.
These structures may be producing a pigment, because I do not remember contaminating with stains. The structures are reddish-orange, and look like they could be attempting to assemble a larger organism.
This does exist in nature, but usually from decimated multicellular life-forms.
Anyway, I did not see these in initial scans, now they are everywhere.
I was expecting cell clusters to form, but the medium is different from the one that supported these cell groups. However, there are cellular organisms developing in this medium as well, and I have posted a micrograph of one of them below.

Continuing with this strange microbe

Small cluster of rosegerms embeded in host proteins


Possible development of complex phase cell structures


Interesting structural development.


Notice the consistency among structures.


Same structures


Look at them closely. These are also absorbing cytoplasmic stain, that accidentally got into broth solution.



All micrographs can be enlarged by clicking on image.




I am following this latest microbe, as described in former postings, because it has a interesting and predictable development in vitro. It is from a chronically afflicted individual, taken from puss sample.
I determined anecdotally, that because the lesion was spontaneous, and without external injury, the same possible agent causing systemic problem may be found in puss.
It turns out, that I did indeed find an interesting microbe amongst the self induced defense proteins. These proteins continue to be processed long after the immune cells have degenerated. It would seem these are the host's natural antibiotics. They even resemble protein clouds formed from penicillin. I can tell they are human by distinct odour [a slight cheese smell]. Obviously these molecules are aromatic in structure.

The curious thing about this sample which is being documented, there are phases which eventually result in the differentiation of cells. I am going to take everyone through this.
The sample was obtained from dried out specimen in petri dish.
It was placed in distilled water with vitamins, amino acids, minerals and butric acid.
Then placed in small tube in incubator.
I did initial scans, which I placed on youtube [see profile page for links].
It showed the extent of biofilm production from activated "host" proteins. The density had increased to high levels, then condensed, and settled.
Now I am posting micrographs of the microbe as it develops, in real time situation.
This way, my predictions can be scrutinized, and seen from other perspectives.

I am calling the microbe "rosegerm", until I can ID it. As seen in micrographs, it starts as a small rose like structure, and progresses to become larger, and has spores being produced in them [it appears so]. As they enlarge, the formation of cell structure appears. In later date, we may see entire cell clusters.
I think this may be a fungal phenotype of microsporidium [but please don't hold me to that]!
I will post every now and again, as the culture matures.

Micrographs of suspect microbes

Here are some interesting micrographs--plus one video of microbes developed from sample of person with chronic problems.
This particular sample was taken from puss, however, the same microbes emerged from blood smear [both samples were treated in process which I developed for assembling problematic microbes originating in body fluids]. I rely on the assumption the microbes utilize advanced system of quorum sensing.
I have my share of critics, I can assure you.

The first micrograph is a mature cell. It makes things, then becomes empty, with a few granules left.

The second photo shows colony of cell types. I don't know what they are, but were assembled from free moving proteins\microbes which start as type of biofilm.
The biofilms are observable throughout solution, but only some become active.
The cells that develop, are varied. Some show protozoan type activity, others move slightly, and others interact. I had attempted to grow these in medium designed for fungi, but nothing fungal grew. I plan to repeat this excercise, with control.

The third micrograph shows more colony cells but shows differences among cell morphology.

The last video, shows the essence of the "bee swarm" clouds I have described as observing. [these are stationary] I have decided this is chemical activity, likely as a defense mechanism. It is reminiscent of mammalian granulocytes.

Swarming germs

Unfortunately, the clip shown is too small for good viewing.
If you look to the far left terminal of the cell cluster, you will see movement. This activity is from microbes being released from cells, where they will join up with others to form hives. From these hives, individual microbes with join up with others in free solution, where they will form swarms.

PCR results are in

I have sent the specimen being documented in this blog, for mtDNA analysis, and the preliminary results are in. It is unknown at this time. They ran queries for fungal sequences and it came up negative. Many, who are familiar with microbiology have suggested it may be fungal in origin. This would be natural assumption, judging by morphology, as seen in micrograph above.
It is apicomplexan, but classification may be a task ahead. The DNA is still available for further sequencing, which may have to be done.
Is it novel? Well, it certainly has not been well documented. Since people like myself are infected with this or variations of this, as a human disease I would have expected a faster hit.

Documenting curious microbe. Pathogen?

Click on image for closer scrutinity


I have isolated a very interesting microbe, and have photographed it through high performance camera at special settings under dark field at 600+-x.
The microbe is motile, and tends to roll. It is made of smaller subunits, which I never would have seen without the computer images. The subunits are green, with red on polar end. I have observed the units break off and migrate away from mother unit. The unit, which I call "sponge integer" has been cultivated from sample placed in 30% bleach with purified sterile water and incubated at 37+ degrees C. The sample was blood obtained from microscopic slide wet mount.
The donor, has multiple acquired symptomologies without definitive diagnosis. Suspicion of bartonella has been noted by specialist, but I do not see any indications of this.

If you study the image, you can see the surrounding subunits, these are found throughout sample. There are also other ameoboid type microbes in the solution, and scattered biofilms.
I speculate the red terminals are acidic molecular residues, and the green is neutral PH. This type of configuration is noted in microbes I have observed with intracellular parasites. I am not jumping to conclusions here, but feel these microbes are worthy of recording for future reference.

More on parasites

I have been trying to upload some important clips, but they would take.

Two cells at high magnification

The first photo is from my "oocysts". The fact these are occysts will be confirmed soon through mtDNA analysis. It is not fungal [unless recombinant], because sporozoits were grown and observed, and even infected other cells---check old posts for documentation on that. Sorry, the photo was taken by commercial camera, but it is high magnification. It is at same setting as second micrograph shown.


This second micrograph is a very high magnification of 2 cells from on going culture of unknown spores. There has been much activity between metabolites, microbes etc., in solution, and the appearance of cells which I say could be oocysts. But yeast cells, and penicillium molds have also been isolated from original source in dry plate culture. I have compared the spores from the {obtained} fungal specimens to objects in free solution broth. From observation, I have eliminated those as being the source of growing bodies in solution, which I believe are parasites.

The cells in micrograph look close to cells I have isolated from my own body, but are from individual hundreds of miles away, whom I have never even met.

Immature microbes, becoming something weird

Click on image for close up
The first micrograph is taken under normal light setting, shows flat view of microbe. [BTW this microbe glides]

The second is under dark field, the light refracts, so there is limited clarity. The colors are very close, though. There is methylene blue in the solution, which causes some of this effect. The energy of the microbe, does emit color. It was seen moving, before capture. They do not live long under glass.

The yeast in question

This was the yeast grown directly from slide sample, although I isolated 3 strains of penicillum as well. The yeast was pure white.
The cells making up the penicillium had some similarities with the growing cells [in pic 3 posts down from here] but the ones in question, seem to be different.
I would have guessed yeast right off the bat, but the microbe in question seems to be directly associated with these cells. I am almost sure they are sporozoites, and if this thought is not swayed, I will suggest to donor, that we have it analyzed genetically.

Check out the suspect oocysts in previous photo's and take note of the cell wall. It has definite thickness--it is a different cell.

More micrographs

These 3 micrographs are as follows: 1) bio film seen as typical film in sample [stained with methylene blue] it appears almost 3D under direct observation, with the stained section in top layer 2) typical bio film, with heavy globulins 3)oocysts[?] which are under investigation--stained with MB. It is not surprising the suspect oocysts stained heavy. This was seen in oocysts from my personal specimen sample obtained from diarrhea contained entirely in mucus.
These oocysts[?] developed from blood specimen of person with chronic symptomology.

Preliminary micrograph of suspect microbe

Click on image for size increase. [4-5 um dark field]

This is just a quick peek at what I "suspect" is a participant in the process which results in the emergence of oocysts. This one is later stage, and there are small proteins tethered to one end.
In the host, the first observation from blood, looked like a rod bacteria. In later experiments, this same ron type bacteria became present. Other forms resembling this form, had proteins which moved in and conjugated with this structure. It becomes covered over time, and starts to produce globulin structures. This is a far as I get.
There are also cysts in same solution, so to much going on for me to make any kind of conclusions.

Click on image for larger picture.

This micrograph is from a sample which contained strange, non-bacterial microbe, which shows signs of complex organization. I have gone through great lengths to promote differentiation of microbe into other stages of development. This compares to trying to make a caterpiller make a chrysalis, and become a butterfly!
Being lead by intuition, some scientific method, and experience, I eventually came up with these structures, which I think are the oocysts made by unknown microbes.
Any comments from experienced microbiologists will be most welcome.

This is a common protozoa to "my" gut. It is big, by comparison to other protozoa and produces cysts. It is hard to tell if these are problematic, because I have only seen attack by leukocytes one time.
The apicomplexan parasites collected previously, have been isolated in mucus, therefore have been associated with immune response, and deemed problematic.
Futhermore, they have been examined in culture for a variety of phases, and been evaluated.

Protozoa

 

This is a protozoa, common to my gut. There seems to be some activity, around the microbe. When the pseudopods extend, it looks like tiny cysts are being secreted. Unsure of that part.
The micrograph was taken by my new camera, and adjusted under different lighting than original view. The detail came out nicely. Click on image for enhanced view.
The background particles are common bacteria.

stuff on proteasome from science mag

Ubiquitin, Proteasomes, and the Aging Brain

Douglas A. Gray, Maria Tsirigotis, and John Woulfe

Douglas A. Gray is at the Ottawa Regional Cancer Centre, Ottawa, Ontario, Canada K1H 1C4 and the Ottawa Health Research Institute, Ottawa, Ontario, Canada K1Y 4E9. Maria Tsirigotis is at the Ottawa Regional Cancer Centre, Ottawa, Ontario, Canada K1H 1C4. John Woulfe is at the Ottawa Health Research Institute, Ottawa, Ontario, Canada K1Y 4E9 and in the Department of Pathology, Ottawa Hospital, Ottawa, Canada K1Y 4E9. E-mail: Doug.Gray@orcc.on.ca
http://sageke.sciencemag.org/cgi/content/full/sageke;2003/34/re6

Key Words: ubiquitin • proteasome • neurodegeneration

Abstract: Ubiquitinated proteinaceous inclusions are the hallmark of many neurodegenerative diseases. Inefficient proteolysis might lead to the accumulation and ultimate deposition of potentially toxic entities as inclusions within neurons or glial cells. This hypothesis is supported by genetic evidence both from patient populations and from engineered mutations in genes that encode ubiquitin/proteasome components in mice. The appearance of similar inclusions in the brains of elderly individuals of normal and subclinical conditions begs the question of whether there is a general age-related decline in the ability of the ubiquitin/proteasome pathway (UPP) to recognize and eliminate abnormal proteins, and whether such a decline would be reflected by changes in the abundance or activity of some or all components of the UPP. Here we describe alterations in the aging mammalian brain that correlate with a decline in the function of the UPP and review the evidence for age-related changes in specific UPP components. These alterations are discussed within the context of prevalent theories of aging.

Citation: D. A. Gray, M. Tsirigotis, J. Woulfe, Ubiquitin, Proteasomes, and the Aging Brain. Sci. SAGE KE 2003

I am leading up to something here, but my time is being sucked into a black-hole somehow. It is these processes I will try and show, that cause neurological problems with those who have stealth parasites\pathogens.

Quorum sensing and "hive" behavior in microbes.

I am writing based on my own experience with this. I know there is a whole science that is based on pleomorphism, but I have been seeing something, I think is unique.
I have covered quite a bit of ground with my own parasite, and have "almost" established, that it belongs to the family of apicomplexia. This is based on stages I have extrapolated by isolation, growth and observation [DNA analysis pending].

I recently observed blood sample of 'subject' who has multiple symptoms including neurological and digestive issues.
Upon first observation of blood on slide [week-old, but sealed] there was evidence of "spore" like objects in the [1-2 micrometer range dark field] sample.
The neutrophils were releasing granules, [granule proteins abundant] and I think there MAY have been NK cells present. I did not see macrophages, other than the neutro-leukocytes. The leukocytes were loaded up with these spores. It is obvious from this, that an immunotolerance has been established to a certain point. The housekeeping cells, however, are in full force trying to control extracellular infiltration of these spores. There were a few bacteria present, but I could not be sure of contamination.

The 'subject' is chronically affected, but still maintains relative health.
This person had western blot for Lyme disease, and was positive for a few bands, which I reviewed, and decided was not enough for solid positive. It did show many elements of bacterial antigens.
I would have like to seen results for protozoan antigens--------and this is where I am going.
I cultivated the blood in saline solution, at body temp. This activated the spores, which turned out to be small short-rod bacteria. They created aggregates or "clusters" which had a very interesting pattern, shown by spectral rings in dark field. Many were independent, and there was an increase of very small proteins. This could have been host proteins, but many more spores were developing.
As these clusters increased, I started to notice [after a few days] yeast-like cells growing in chains of 4. There were singlets, triplets, quins, and more, but the vast majority were quads.
These cells would eventually migrate together, and create a larger unit, something like the smaller bacterial looking things were doing.
This reminded me of the behavior I had observed with my oocysts! I therefore believe these are indeed oocysts. But the morphology is remarkably phenotypic of yeast cells.
The sample does not grow on plate nutrient with sugar, and microbes are temperature sensitive, going into dormancy outside 37 C.
I am speculating, there is a fungal quality to these microbes, as well as protozoan. The small microbes, look and behave like my own 'zoites.
I will be analyzing the spectral rings, and comparing them to my 'zoites with special equipment soon.
I think we have another example of these mystery apicomplexan-like parasites.
More tests are now being conducted, and I will post more on this later.

Proteasome and it's role in immunity

It is one direction scientists are taking to review some of the aspects of disease.
One very important mechanism of antigen presentation, is the digestion of said antigen, into small [8-10kDa] peptides which fit well within the Class 1 MHC receptor pocket.
Since everyone has different genes that code for MHC receptor, there is a significant polymorphism that exists here. This is how nature ensures greater diversity among same species, so odds of survival are increased in the face of danger from disease.
If one person cannot handle a solid presentation of antigen because of poor binding properties of antigen, another person may do this extremely well. This individual will have best chance at eliminating dangerous antigens [virus, intracellular disease etc].

The proteasome is a little molecular machine in all eukaryotic cells which digest proteins within the cell for recycling, antigen presentation, or elimination.

I have to make a personal comment on this process. Why can't we grow cells in culture, of people who have ability to digest certain antigens, isolate them, and serve them to a person who has poor ability to digest this particular antigen.
It would stand to reason, the individuals t-cells will pick-up these pre-digested antigens, and have far better chance of making a successful presentation.
Why could we not experiment with the proteasomes of certain frogs or other exotic life forms to evaluate the antigen digestion abilities of their proteasomes, and keep a data base of these records?

Potential for creating unique vaccinations seems promising using this direction.
That was just a personal observation, and maybe it's old news. I have not researched it very far, yet.

Can protozoa or apicomplexia shift to fungi forms?

This is something of interest to me. I have no evidence of it, except the fact that if it did happen, and if it only happened within specific host or primary host, it would be missed by observation. Because these microbes carry such complex gene-line variation, it can be hard to recognize mutations, and related phenotypes.
Plus, what about gene sharing? Some microbes are known for this, and some share plasmids which can offer extended genetic characteristics to the microbe.
I will be posting more on this later..........

Medical experiment

I am taking an immunological approach, strategically designed to prompt neurotransmitter shifts in the body, directed at disrupting mast cell stimulation.
This will be attempted by ingesting 120 mg of pseudoephedrine hydrochloride and 7200 mg of ascorbic acid at once before bed.
This is only a preparatory experiment to record physiologic affects.
Pathways of interest are those involve with alpha melanocyte hormone regulation, numerous cytokines and other regulators.
Chemical competition for mast cell receptors is a key goal in this experiment.

By disrupting the status of chronic stimulation of mast cells, I hope to damage the equilibrium, infecting {apicomplexia} parasites are creating with immune functions. I am assuming the parasite is deliberately sequestering the mast cells to control mediators governing other immune factors.
The protocol may not be effective, but observations could be useful.

This is March 15 2009
Day after the experimental drug\vitamin C cocktail;
My symptomolgy dropped by close to 80%! This was completely unexpected, but confirms the hypothesis that mast cells are the central area of concern. This places the disease in awkward direction.
It has been proven the parasitic 'zoites infect these cells in vitro, so elevation of activity with these cells is no surprise. The consequences of sustained amplitude in cell activity and differentiation, is physiologic toxicity. This is now an established fact based on solid scientific evidence.
The next hypothesis is; does the parasite deliberately create a system of survival by using the mast cells and\or other highly specific immune processes to it's advantage?

The fact these parasites cannot live among lactobacteria in vitro, and my digestive system is almost devoid of these types, suggest the mast cell activity kills these bacteria which could be advantageous to the parasite. The epithelial cells are very active producing cryptocidins as well, which might also play a part. It seems these molecules in high abundance, help growth of parasite in vitro. It also has antibiotic effect.
I have noticed high defensin activity in prostatic fluid, and cysts have been observe there [I have seen other unidentified objects of microbial interest there as well].

The next phase of this experiment, is to triple the dosages{per day}, add charcoal and chorophyll to the protocol, and dramatically increase probiotics, and yeast to diet.
I will do prostatic fluid exam in one week. Stool exams will be on going to observe changes in microbiota.
WARNING Do not attempt this experiment. There are dangers associated with cardiac functions in some people.

Date March 17\2009
The experiment has been successful in making a dynamic shift in my overall physiology, symptomology. The symptoms which have been undeviating for many months have shifted.
I think the ascorbic acid has interacted with the pseudoephedrine hydrochloride more than I had expected.
There has been changes in sleeping patterns, and big changes in digestion.
Mood changes are in good territory, indicating hormonal\neurotransmitter adjustment.
These are not subtle changes, and are related to the protocol. Further investigation of these chemical interactions must be elucidated.
Note: I have reduced the pseudoephedrine to 60 mg and ascorbic acid to 500 mg once a day. I have added bentonite and chlorophyll to the protocol as well.

March 18 2009'
The reduced dosage of pseudoephedrine hydrocholide, has allowed homeostasis functions of the body to become stable, and therefore effects of anti-inflammation to wear off. The ascorcic acid, also speeds the metabolic processes of the pseudoephedrine hydrochloride, through the body, and reduces affect.

The impact of infection was not affected, and symptoms are very quick to return.
Therefore, it would seem decreases in mediators from mast cells were either not strong enough to cause differential impact on parasitic factors, or parasite has no intimate reliance on mast cells specifically.
Antigen stimulation, at sub-levels of immune activation are what probably keep symptoms and parasitic infection in balance. ---end of study-----

Can parasites manipulate thoughts?

Parasite-induced modification of animal behavior, is one of the least understood phenomenon of parasite\host relationships.
There are plenty of clinical studies, many focusing on animals and insects in the natural environment.
There have been studies on domestic animals, and on humans. Plenty of scientific data exists on the subject, but it still remains a shadowy area of research especially where human behavior is concerned.

Toxoplasma gondii has been characterized as a parasite capable of inducing behavioral modifications of intermediate hosts. The exact mechanics of this process is still being evaluated.
Many larger parasites like worms, [which are animals] have rudimentary nervous systems, and therefore can secrete hormones, neurotransmitters, and many other biologically active soluble proteins. The potential for direct, or indirect physiological and psychological affects on the host are obvious. The wastes produced by these organisms further add to the complexity of this interconnectedness.

In many cases, host behavior modification by parasites have been seen as an adaptive response to benefit the parasite by increasing it's probability for successful transmission.
It would seem an evolutionary process is active within the host, for purposes of survival.
In order to reason with the mechanics involved in host manipulation, one must first examine the biological parameters confronting the parasite.

First, the energy spent on manipulation of host, will cause a deficit of energies spent on other functions such as growth.

A theory that governs parasitic manipulation effort on host behavior goes something like this:

1) typical same species population size decreases
2) prevalence increases
3) longevity of parasitism within host decreases
4) passive transmission decreases
5) potential reproductive capacity decreases

To access some of the some of the physiological responses associated with parasitism, clinical studies have focused on some of the following areas: neurology, endocrinology, neuromodulatory and immunomodulatory components and cognitive studies.

There are many intriguing examples in nature, that highlight the incredible evolutionary sparing between parasite\host dynamics which can determine and alter destinies of species populations.
These are important observations to consider, when evaluating the circumstances of human pathology.

In my next blog, I will focus on some clinical examples of host manipulation from a cross-section of human parasites.

Clinical test modalities for toxoplamosis

The current epidimiological estimates of latent toxoplasma gondii infections world wide are set at approximately 30%. The rate among eastern European countries is near 80%.
I have to question the accuracy of these numbers, because it calls into question the rate of pseudopostive test outcomes. There is also the question of affinity and avidity of antigen antibody complexes in these tests, which weigh heavily on qualification data.

In essence, I think the seroprevalence of toxoplasma species among epidimiologically significant study groups, may be largely inaccurate.
Furthermore, there may be other homogeneous apicomplexan species infecting humans, that go undetected because of over emphasis on toxoplasma gondii spp.
For example, patients who exhibit symptoms of so-called Lyme disease, are very good candidates for these types of parasitic infections.
I would like to discuss some alternative tests, that will encompass an expanded range of species, and maintain accurate quantitative and qualitative results. These tests will have to be cost effective for routine clinical analysis.

Circulating antigen assays are the best, efficient and cheapest method for detection of toxoplasma and related apicomplexan species. {edit: includes non-antigenic proteins and foreign DNA\RNA residues} Chinese studies, have found the rSAG1 gene, in truncated form, can be easily detected in urine of newly infected and chronically infected mice. The antigen can be manufactured through expression vectors [e. coli, yeast, baculovirus] and made available for urinalysis assays. It is designed for specificity against Toxoplasma, but covers numerous strains.

PCR testing has pitfalls, mainly due to the high sensitivity of testing procedure.
Problems such as inhibition in sample, laboratory contamination, and skill of operators\technicians are often encountered.

My personal opinion is based on the above. I say finding mitochondrial antigen markers that cover a wide group of apicomplexans should be the focus of researchers in the field. Development of affordable testing motifs should be a priority, given the rate of unexplained climbs in chronic diseases that go undiagnosed in North America, and around the world. The importance of this parasite as a disease, must come into the foray of modern medical consciousness.

Discovered novel human apicomplexan parasite

I am making a claim on this parasite, but mtDNA COI barcode will be needed to do this officially.
I have discovered the connection between my gut infection, and my prostate infection. This was essential to do, before making this announcement.
It shows the ability for the microbe to migrate in the body, and infect cells.
The macrophages of prostate pick up cysts, and cannot digest them for antigen presentation.
The cysts seem to develop into sporozoits directly as a single sporozoite. They are not direct relationship to shedding gut oocysts. The macrophages eventually end up in gut, where presumably, the cysts
become active. The genome of this parasites is very diverse, and the various stages and forms are difficult to track.
This parasite in other forms or strains could be a factor in many chronic health diseases.
One diagnostic indicator, is elevation of mast cell activity.

I have included a small clip of the sporozoite to show one form and stage of growth. There is a distinct feature of this one, a small finger-like protuberance which acts like a probe. It could also release a spore, but I am not 100% sure about that. The crystal is rhodamine 6G dye, and there is background debris. The image was taken under phase contrast at high magnification. It is the orange rod-like object in middle of frame.

Challenges of live microscopy

I have just completed the observation of prostatic fluid, incubated at 37 C for 24 hours.
The object of this task, is to find any signs of apicompexia in the sample. The sample is much like surum, in that the contents contain many immune cells of haemotopoietic lineage. As mentioned in previous post, the cells are lymphocytes, monocytes, neutrophilic granulocytes, monocytic granulocytes, and mast cells.

The cells in the sample, were almost all denatured due to extremely active proteolytic proteins, defensins etc.
Evidence of parasitic infection, was proven at this time. There was active sporozoites present, with typical morphology. They were lancet-shaped, and banana-shaped, and some were deformed. The light reflection was very high, indicating tough or hard-like membrane. They had distinct, but low motiltiy.

Back to problems with live specimen. The sporozoites died quickly, from abrupt change in environment [presumably].
Several active life forms were found in specimen, which shared similar characteristics, and have me struggling for clear explanation.
Since the whole exercise is to find co-relationship between intestinal findings and prostate findings, my personal bias conflicts with my scientific objectivity. I am expecting to see direct morphological similarities, but this is not entirely the case.
This is what I observed:
Some of the orb-like opaque objects seen previously in\on macrophages of fresh specimen, have survived the lytic processes. Now they are maturing under glass, and "budding" exactly as yeast would. Some become very large, others are small, but both types will bud. If I had not seen this with intestinal oocsts, I would automatically consider yeast as my first guess.
There are no difference between looking at these, and looking at natural air bubbles, with the exception the live ones move. There is observable movement of small object inside the orb, moving back and forth within membrane. No other organelles are observable. When the object starts to bud from membrane, it retracts and moves about the membrane causing distortions. It is almost like something in an egg, trying to find a way out.
Other more advanced growth developments show the new bud, at one third size of mother cell, connected by a membrane. This is very similar to observation of intestinal oocysts.
The sporozoits, share same light frequencies so I am suspecting, the sporozoites emerge as singular unit from these "orbs". I don't have solid proof of this yet. I have seen these forms directly in live stool samples

There is something else. Small cell-like bodies emerged everywhere. They are dark, almost black and measure about 3 um [I am using phase contrast at full mag enhanced]; they look like another reproducing cell-type. There is obvious nuclear movement within cell, and it also produces bud, and orbiting granule, attached by small filament which is retracted back into cell. It has no light reflective qualities. I must question if it is residual host stem cell activity.
The UV absorbs some of the opaque orbs, not sure what this indicates [maybe tyrosine content].
I may attempt to place some images later.

Testing antigenic reaction in mast cells

I have a condition called prostatitis. The cause is officially unknown. I have suspected the cells in prostate were infected by the same parasite as intestine, but have a hard time quantifying microscopic analysis, due to large number of cells present in prostatic fluid.
Today, I did a test to see reaction of prostate cells to parasites stored in vitro.
I have the original sample which contains oocysts, and I have a culture of the sub units which are the infective agent I call 'zoites[proven by infection of cells found in saliva].
The procedure goes as follows: stimulate prostate gland using traditional method. Collect fluid on glass slide. Use dropper and isolate small specimen to incubate in vitro for later testing. Use the dropper to prepare three slides.
In the first slide, place 'zoite containing supernatant next to prostatic fluid on same slide.
In second slide, place first sample containing oocysts next to prostatic fluid.
Third slide remains as a control.

Results:
In the first slide, the 'zoites circulated among cells without eliciting any significant granular reaction from native cells.

In second slide, the oocyst containing specimen immediately activated all cells violently.

The third slide showed neutral reaction.

I have not determined that the cells are majority mast cells, same as found in saliva & gut. The nucleus is ill defined. There are some macrophages of monocyte and neutrophil lineage in sample.
The disturbing thing is; there are lymphocytes scouting surface antigens of mast cells.
Many cells are looking ruptured, or distorted. This should be indication of granular release [I hope]. There are also cells which resemble protozoal stage of parasite, and oocysts in control. These structures are different from oocysts found in stool sample [if they are oocysts] and are same size as lymphocytes. It is very difficult to tell them apart. The spheres have high amoeboid-type movement, and protuberances which cause distortions.
Many macrophages have opaque spheres in or on cell membrane which are not part of natural granules. Parasite in other form[s] may be in specimen.
I will need an antibody marker to confirm this.

As you can see, the indications are not good. The parasite may have antigenic immunity in certain stages. The cells in prostate seem less sensitive than cells found in saliva.
The connecting factor is the presents of elevated levels of mast cells.
It could unify some theories on causes of chronic disease if these type of parasites are more common than is known. If say, toxoplasma infects mast cells, it would be difficult to detect among granules. The implications of this are quite significant, given the mediation capabilities of these cells.

More on blood sample

In a previous post, I described the examination of blood from subject having MS like symptoms. The last round of cultures display fungus of some sort. I have obtained this from plate cultures, and a broth culture under incubation at body temp. 37 C.
I had both plate and broth [controls], both are negative for fungal growth.
Not sure if this is relative, but earlier observations of slide preparation before testing, showed very small objects thought to be viral. I am now contemplating if they were indeed fungal metabolites. The product was too small to be spores.
Since the subject was positive for aggressive mycoplasma, and possible L form bacteria, I wonder if the fungal products are connected, or if it is a natural occurrence.
I will have to repeat the testing with my own blood to see if I get a similar result.

Mast cells and chronic symptomology

Mast cells originate from haemapoietic precurser cell expressing CD34. It is proposed that they evolve from monocyte lineages. They are morpologically round, about 5-10 um in diameter and have densly packed granules, often obscurring the nucleus, which is about one third the size of the cell.
Mast cells are intimately involved with host defense and wound healing, and many other things.
There are primarily two different classes of mast cells; one is distributed in connective tissue, and the other in mucus membranes.
Mast cells have a very broad range of functions, and have a very diverse role in the immune system and other physiological events.
Mass cells are directly involved in the inflammatory process.
The granules have extreme biological activity, and can mediate a wide range of physiological events when released. Degranulation is stimulated by direct stress {such as injury} or by cross linking with IgE receptors and activation of complement.
I propose, that other soluble peptides, and neurotransmitters can also act as activation factors.
Mast cells present a high affinity receptor for IgE [Fc region] called FceRI.
Important to note, that mast cells have recently been attributed as defense system against parasitic infections such as intestinal worms.
Here are some of the known characteristics of granule mediators:
Histamine, proteoglycans-- mainly heparin {anticoagulant}, serine proteases, lipid mediators-{eg. eicosanoids}, prostaglandin d2, luekotreine C4, cytokines and chemkines.
They also contain serotonin, dopamine, trypase, and chamase.
Mast cells secrete many pleiotropic {multiple effects} cytokines. This means there may still be many functions of this cell yet to be discovered.
Because of the ubiquitous distribution of mast cells throughout the connective tissues, epithelial surfaces, and close proximity to blood vessles, it make their products available to a large variety of cell types including fibroblasts {like skin making cells}, glandular epithelial cells, nerves, vascular endothelial cells, smooth muscle cells, and finally cells of the immune system.
Plus, there is a myriad of lipid mediators [important to note], proteases, proteoglygans {which can also play a role in the activity and stability of proteins and signaling molecules}, and again, cytokines and chemokines.
There is new evidence that mast cell cytokine responses are regulated by pathogens\parasites and their products. This is a very important point to remember, when considering the potential symptoms attributed to chronic infections. So, in other words, mast cells can induce cytokine activity relative to the type of antigen or pathogen.

Examining blood sample of subject

I have looked at the blood sample of a person with MS-like symptoms including occasional loss of feelings in limbs among other things. He has had MRI, other scans, and multiple tests. Nothing conclusive.
The sample was old, but sealed. I have the person do this so as the cells die, they cannot dry out. This often prompts the endobionts and stealth bacteria to emerge.
I check this out using a variety of microscopic techniques.
In this case, I have found a number of "balloon" type structures distributed amongst the cells. There were a few lymphocytes present, and one had attached to a "balloon".
This indicates to me, an antigen\receptor interaction going on, telling me the entity is dangerous, or least unwanted.
I usually assume at this point, there may be a cell-disease agent in subjects system.
Cultures are prepared with controls, to see if any further information can be determined.
I this case, there was a bacteria which grew, but had to be destroyed. I believe it must have been a contamination.
The conclusion after multiple tests; the primary bacteria is mycoplasma. This particular strain was aggressive, and pleomorphic. There were very distinct morphological characteristics which give me confidence about the conclusion.
Antibiotic infused liquid culture revealed there was complete inhibited grow of bacteria.
Tetracycline was used for this experiment.

Securing indentification of ambiguous microbes

Today I want to talk about the options I am considering for proper identification of my parasite.
This is important, because there is no doubt that this organism is important in the investigation of digestive and other problematic human health issues.
It is also advantageous for developing testing modalities.

The best test for identifying this microbe is called "COI Barcode" [cytochrome oxidase] which is a protein coding region of mitochondrial DNA {mtDNA}.
The area of the gene, the first half containing approximately 648 base pairs, is the easiest to process thus the cheapest.
Here are 3 main reasons to use COI barcode.
1) easiest to recover, including degraded sources from diverse taxa
2)readily aligned for sequence comparisons
3)effective for making distinctions between closely related species including both vertebrate and invertebrate taxa.

Six practical reasons for using this barcode:
1) Works with small traces. Barcoding can identify species from "bits and pieces" including material in foodstuffs

2) Works for all stages of life. Barcoding can identify species in all it's many forms, from eggs and seeds, through larva and seedlings to adults and flowers.

3)Unmasks look-alikes. Barcoding can distinguish among species that look the same, uncovering dangerous organisms that look the same as harmless and enabling a more accurate view of biodiversity.

4) Reduces ambiguity. A barcode provides an unambiguous digital identifying feature for quantifying species, supplementing more analog gradations of words, shapes and colors.

5) makes expertise go further. Scientists can equip themselves with barcoding to speed identification of unknown organisms and facilatate rapid recognition of new species.

6) Democratizes access. A standard library of barcodes will empower many more people to callby name, the species around them.

Experimental success!

I have managed to demonstrate the infection process of 'zoites into cells which were grown in saliva.
To re-cap; I placed saliva in glass tube to grow cells. I assume they are epithelial type cells. They tend to be designed to secrete huge amounts of antimicrobial proteins. In any event, they must represent similar cells in mucosal secretions throughout bowel.
The proximal region of the small bowel, is the assumed area of parasitic infection of host [me].
The latest observation shows complete infiltration of some cells. This seems to indicate a specificity for cell type, or some discrete characteristic of particular cells. Internal protein activity of some of these cells are indicated. The activity of infective 'zoites seem to calm once positioned within cell body.
I have posted a small clip showing total infection of cell, with active protein still observable. The larger reddish particles are the 'zoites {still don't have proper term, sorry} and the small particles showing Brownian movement are the active proteins.
The clip is shown at 600-700x under dark field conditions.

Results from experiment

I wish these microbes were easier to predict. The anticipated infection of cells was not accomplished as expected. There were a few parameters in the experiment which may have altered the outcome.
First, the chemicals in the specimen supernatant [bleach, lysol disinfectant] had affects on the saliva derived cells. The cells massively amplified production of antimicrobial proteins.
The initial observation, before incubation showed 'zoites infiltrate cells. They did this slowly, by attaching to surface of membrane, and eventually slipping to the interior portion of membrane. Then, proceeded to central part of cell.
Predictable you say?
The incubation mix, showed significant drop in number of 'zoites. The amount of protein products from cells, increased a thousand fold. Many cells were lysed, and the cells that were infected, had active granulation which was making life difficult for the 'zoites.
I conclude, that these cells are not suitable housing for the 'zoites but that cellular infection is definitly the objective of these microbes.
It also demonstrates why I have problems with dehydration, and depression of eletrolytes. The cells use up salts, and other resources for the constant generation of cells, and their chemical products.
The million dollar question is; what cells are being infiltrated, and which defense mechanism is showing ineffective action against these 'zoites?
I have included a short "clip" showing infected cell, in conflict with intruding 'zoites. This is at 600-700x mag under dark field conditions. The visual information is not the best [waiting for expensive Lumenera camera to arrive], and there are no annotations. You might have to squint a bit. The 'zoites are orangeish red, and the background movement is chemical action from cell. the activity is observed at lower
left of cell.

My apologies, the transfer of clip was not as clear as expected.

Experiment to infiltrate cells with 'zoites

If you place saliva in a test tube, cells will continue to reproduce. These cells will protect themselves against invasion from microbes through TLR's and the production of basic antimicrobial proteins. They need very little attention to conditions governing growth.
Since the parasitic 'zoites [term used until formally classified] seek out cells for housing, and cannot survive antibodies, I think these cells should provide the perfect model for examining the interaction of the cell invasion process. I will place the infective agent into small tube with cells, and incubate for 12 hours. I will report in next post, with photo's.

Starting a small research lab

Having done microscopy work for 5 years, and investing much time and money studying my own fluids, I have decide to extend my research practices to include samples from other subjects.

I have already done this on a small level. I feel there is much that can be accomplished by running routine, as well as complex tests on subjects who complain of chronic symptoms. Subjects must have been previously evaluated by standard medical tests, which have failed to elucidate any obvious problems.
Some of the tests I would like to do are as follows; HLA determination, cytokine tests, blood morphology in native samples, antigen screening of intestinal fluids and urine, peptide screening and documentation, protein isolation from aforementioned fluids and development of data base of random protein samples, and toxicity tests for body fluids to name a few.
I would like to venture into hybridoma technology [some day] and do work with monoclonal antibodies.
There are many things to consider when designing a lab.
First, you must have an appropriate location, there must be equipment and supplies, modifications like vent-hoods, disposal protocols, safety protocols, lab wear, incubation station [vents] etc.
Since I am doing this on a small budget, the location will be quite small.
I plan to have a person in the lab doing work everyday, so students will need to be considered, and small compensation awarded. This will be stressful on budget, so donations to the lab is a must.

Therefore small advertising campaigns and circulation in health-concerned events to distribute business cards, and generate interest must be part of my job.
The idea, is to build a facility which is interactive with ordinary people. This is a way to help individuals with their own research and give them vital information they need to help themselves.
Another feature of the lab, will be consultation.
I would like to share the space with a health expert such as nutritional consultant. This way, there can be co-operation between different modes of information.
The key feature of the lab will be high performance microscopy, and micro graphic documentation. I plan on using this feature to collaborate with professionals in the field, who will receive a fee for reviewing micrograph's, other data.
I would also like to have a network of other labs on file known for specialties in various disciplines.

Understanding the behaviour of my parasite

One of the interesting characteristics of my coccidian parasite are the mother cells. They "bud" which I find unusual. These are seen here to the right. The twin oocysts [not seen here] are opaque and contain spores only [gamatocytes?]

All attempts to transfer the original oocysts to fresh medium have failed. I have used many different mediums, and have settled on purified beef liver extract with 30% bleach, and 20% lysol disinfectant. This medium could not sustain the oocysts for long. However, the bradyzoites [for lack of term] multiplied to fill the supernatant significantly. Bacteria have been controlled by addition of above mentioned chemicals.
A mystery exists, because the original sample still sustains "twin" oocysts, and bradyzoites have settled into nests without over contaminating rest of solution.

Understanding the nature of coccidian parasites

From studies done on plasmodium and toxoplasma gondii to name a few, I find intracellular parasites are a marvel of nature. They can literally cause subtle genetic manipulations in host physiology, and in some cases psychology. My own microscopic observations, demonstrate this parasite has a complexity beyond many microbes in nature, and it's survivability will ensure it's place in human epidimiology for some time to come.
I am looking for other scientists or even hobbiests interested in these microbes, for continued discussion and exchange of scientific data.
I myself am infected with a unique type, and will dedicate the rest of my life to understanding this parasite. I have microscopic data available for viewing, and will post them on occasion.
That is it for now,
umm