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.