Morphology
   
Life Cycle
   
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The Nurse Cell
Parasite Complex
   
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Summary Gallery
 
Biology of the Nurse Cell — Parasite Complex
   
All information contained in this web site relates primarily to Trichinella spiralis, unless otherwise stated.
For a light-hearted look at the process of Nurse cell formation see: Trichinella spiralis: Growing Up Parasitic, the Early Years

I. Morphology of the Nurse Cell

Gallery:

The Nurse cell is a unique consequence of the host cell’s association with the infectious L1 larva of Trichinella spiralis and other Nurse cell-forming species of Trichinella (T. britovi, T. nelsoni, and T.nativa). It presumably functions to nourish it as well as protect it from host immune responses. The mature Nurse cell is morphologically distinct from any other mammalian cell type; no other pathological condition induces such a radically different, and yet functional cell. The Nurse cell-parasite complex can survive in the human host for up to 30 years, and in most other species of mammal for the life span of the animal. For this to occur, the worm must immuno-suppress the host, yet nothing is known regarding the mechanism(s) employed by the parasite through which it keeps the host from killing it. Repeated exposure to infective L1 larvae via the oral route somehow breaks down worm-based immuno-suppression and larvae from prior infections are killed. With enough repeated infections, the host is rendered sterile with regards to trichinella.

Intravital microscopy has revealed the relationship of the Nurse cell to the surrounding tissues. The following video is an in vivo, living Nurse cell-parasite complex . The Nurse cell begins to form the moment the newborn larva penetrates the striated skeletal muscle cell.

Schematic of a Larva Invading a Muscle Cell

BEGIN SPECULATION
The mechanism(s) by which the newborn larva enters the host cell is not known, but morphological evidence gives hints as to the overall process. In muscle tissue, the larva penetrates out of the capillary and then braces itself against the adjacent muscle cell. In doing so, it gains leverage enabling it with its anterior end to depress the sarcolemmal membrane to the point of breaking. Then, it most likely brings its stylet into play, poking a hole in the membrane, causing the host cell to "explode", witness the "ragged" edges of the sarcolemmal membrane observed just moments after the larva entered the host cell. We envision the overall process as similar to one of us poking our index finger straight into a fully inflated balloon, then somehow managing to unsheathe a sharp finger nail, bursting the frail object. Therefore, we do not think secreted enzymes are necessary to aid its passage from the blood stream to the intracellular environment of the muscle cell. However, this last idea needs to be tested.
END SPECULATION

 

 

 

 

 

 

II. Nurse Cell Formation

Larva enters muscle, muscle proteins
"leak" into bloodstream
Mitochondrial Damage
Secretion of Tyvelosylated Proteins
DNA Sythesis and Nuclear Enlargement
[Documentation]
Collagen Type IV Synthesis
Collagen Type VI Synthesis
Induction of VEGF
Angiogenesis

There are at least two distinct phases to the overall process:
1. De-differentiation of the muscle cell
2. Re-differentiation of the muscle cell into the Nurse cell

The morphological events resulting in the formation of the Nurse cell-parasite complex occur over a 14-16 day period of time after the worm has entered the muscle cell. The diagram below summarizes the overall change.

The accompanying toluidine and methylene blue stained light microscope sections and electron micrographs are from synchronously infected mouse muscle tissue embedded in plastic, and correspond to the time points in the diagram. Click here to see these sections.

 

 

 

 

 

 

 

 

1. De-differentiation of the Muscle

Gallery:

The rate of increase in the worm’s volume is a crude measure of worm growth (see Growth Curve below), and differentiation of its tissues, for example stichosome, is correlated with the growth and differentiation of the Nurse cell. Only the rate of growth of the worm has so far been documented. The larva grows during the first day after entering the muscle cell, doubling its volume. The parasite remains the same size over the next two days, resuming growth on Day 4. During this pause, the host cell becomes disorganized with respect to its contractile elements.

The first five days of the process of Nurse cell formation involves a loss of normal structures and contractile functions. Actin and myosin filaments begin to disappear and the sarcolemmal membrane becomes separated from the contractile elements. Click here for a series of EMs that document these observations. Muscle mitochondria become vacuolated and ATP synthesis is uncoupled from aerobic metabolic pathways beginning on Day 3, and remains so throughout the infection.


 

 

BEGIN SPECULATION
We believe that mitochondrial dysfunction is essential to the life of the developing parasite, since the larva is an anaerobe.
END SPECULATION

Thereafter, up to Day 19, the parasite increases its volume by 39% each day, and while growth continues, the parasite achieves infectivity on Day 14-16.

BEGIN SPECULATION:
We hypothesize that the newborn larva induces at least some of the early events in Nurse cell formation, and may do so by secreting substances into the milieu of the muscle fiber. The anterior half of the newborn larva contains a row of cells inside of which are found numerous granules reminiscent of those seen in the fully developed stichocytes of the infective larva. If these granules contain secretory proteins, then newborn larvae most likely releases them into the newly penetrated muscle cell.
END SPECULATION

 

2. Re-differentiation of the Muscle

Gallery:

Most of the important changes related to down regulation of the fully differentiated host cell occur by Day 8. Enlargement of host cell nuclei are at their maximum on that day (below), and it is at this point in the infection that the stichocytes begin to produce and secrete tyvelose-decorated proteins into the milieu of the infected host cell.

Hypothesis: Secretions of the larva induce host nuclei to enlarge
Tyvelosylated parasite proteins (at least 4) are found on the epicuticular surface (Despommier and Kajima, 1969; Almond, et al), within the cytoplasm of all stichocytes, and within the cytoplasm and, most importantly, the nucleoplasm of all nuclei of the developing Nurse cell. Nurse cell nuclei decrease in volume on Day 10 and remain at that volume thereafter.

BEGIN SPECULATION
The larva must influence the growth of the host cell. Are secreted proteins involved? If so, are any of them related in function and/or structure to known mammalian cell growth factors? The enlargement of host cell nuclei is correlated with the presence of secreted parasite proteins within the nucleoplasm of each enlarged nucleus. The enlargement process results in very large nuclear pores. Does this then obviate the need for nuclear localization signals from parasite secreted proteins so that they may enter and exit the nucleus at will?
END SPECULATION

 

 

 

 

A. Collagen Capsule Formation

Gallery:

An acellular capsule begins to form outside the Nurse cell beginning on Day 10. It continues to thicken until Day 26, and is largely composed of collagen type IV and VI (Polvere, et al, 1998). Collagen synthesis occurs within the cytoplasm of the developing Nurse cell and is directly correlated temporally with the thickening of collagen along the outer surface of the Nurse cell.

BEGIN SPECUALTION
The induction of the collagen outer capsule is a direct result of exposure to secreted proteins of the parasite.
END SPECULATION

Through the use of anti-collagen antibodies and specific RNA probes, the types of collagen present in the capsule were determined. Two dominant collagen types were detected, type IV and type VI. Northern analysis and in situ hybridization studies showed that type IV collagen synthesis begins on Day 10 and ceases on Day 26, while collagen type VI begins on Day 10 and continues throughout the infection period.

BEGIN SPECUALTION
If collagen synthesis is up regulated by secreted proteins from the larva, then more than one parasite protein must be involved, since each collagen gene family is regulated separately.
END SPECUALTION

Type IV collagen is a large molecule (Show structure and give MWT) and is found throughout the vertebrate body, and most likely functions as a scaffolding element, holding cells in various tissues together. In contrast, collagen type VI is a relatively short molecule (give structure and MWT), functioning to bind larger collagen molecules together by cross-linking them, like the individual rungs of a ladder that together support the two legs.

The Nurse cell cytoplasm is infiltrated with numerous, as yet unidentified, mononuclear cells (ems - 6 to 8 -, and light microscopic photos). The number of different types of host cells that gain entrance to the Nurse cell has yet to be determined. Their function(s) in Nurse cell formation and/or maintenance remains enigmatic. Initial observations indicate that the process of cell recruitment into the Nurse cell is on-going.

BEGIN SPECULATION
Careful visual inspection of the capsule by either light or electron microscopy does not suggest that there are any obvious weak spots or holes in its outer surface, although we are not aware of any a systematic search for such breaks. This aspect of capsule formation and maintenance needs to be thoroughly investigated. Since trafficking of host cells in and out of the cytoplasm of the Nurse cell appears to occur continuously after Day 10, the process most likely requires that invading cells break down at least a small portion of the collagen capsule in order to facilitate their entrance. If this is a valid way of envisioning the process, then repair of the capsule would, by necessity, also be an ongoing process. Our model would thus explain the need for continuous synthesis of collagen type VI. In contrast, type IV is a more loosely packed molecule in the capsule, at least it appears that way on electron micrographs. Thus, cells might be able to crawl in and out of the cytoplasm without disturbing its lacy network of fibrils once they have disconnected type IV molecules from their cross-connecting type VI helpers.
END SPECULATION

Suggestions for experiments that explore some of the possibilities raised above:

  1. If various sub-sets of mononuclear cells from the host were first isolated from the blood of an infected animal by cell sorting, then permanently labeled, they could then be injected iv, one sub-set at a time. Labeled sub-sets of mononuclear cells arriving at the surface of the Nurse cell could then be identified and tracked throughout the Nurse cell cytoplasm, based on their marker. The marker has to be internal, since surface markers are likely to be shed during the act of diapedesis.
  2. Exposing an infected host simultaneously to labeled amino acid precursors of collagen type VI could facilitate research into whether or not collagen type VI was involved in any repair mechanism using autoradiography. Labeling of type VI collagen would be achieved by giving label after Day 30 of Nurse cell formation, thus eliminating the chance of accidentally labeling type IV collagen. Patchy distribution of labeled collagen type VI on the capsule surface of a mature Nurse cell would constitute positive evidence for repair events, and may even allow for the identification of the cell that caused an event to occur if marked cells were used simultaneously
  3. Nurse cells can be isolated and maintained for long periods of time in vitro (photos). The potential thus exists for placing various mononuclear cell types in the presence of mature Nurse cells to explore the possibility of observing the process of cells going in and out. Since cells loose their surface markers while inside the cell that they are travelling through, by collecting mononuclear cells after they exit from the Nurse cells in culture, one could possibly identify their surface markers after they are regenerated using specific monoclonal antibodies.

Data Summary Table For Capsule Formation

Click here for the
histochemical stains
  Click here for the immunohistochemistry
         
Click here for the in situs hybridizations   Click here for a
Northern Analysis
 

 

B. Angiogenesis (Circulatory Rete Formation)

A network of circulation develops immediately adjacent to the collagen capsule (PHOTO of vascular casts and diagram), beginning on Day 7, as evidenced by the induction of vascular endothelial growth factor (VEGF) within the confines of the developing Nurse cell (in situ.). The rete consists of flattened, tortuous vessels, some of which end blindly. They are generally wider than capillaries, and resemble vessels constituting sinusoids. The presumed function of the circulatory rete is to enable the parasite to obtain nutrients and facilitate the transport of small molecular wastes. This supposition needs to be documented by experimentation.

Suggestions for experiments to elucidate the function of the circulatory rete.
(see summary of inhibitors)

Interfering with the formation of the circulatory rete seems like a reasonable first — order strategy to approach the problem of the function of the rete for the parasite. Anti-angiogenic drugs and antibodies against essential growth factors such as vascular endothelial growth factor (VEGF) and alpha V beta 3 integrin can be employed. Antibodies directed against VEGF are effective in preventing the growth of capillaries towards the source of VEGF. Examples of anti-angiogenic drugs include fumagillin, that inhibits division of vascular endothelial cells, lavendustin-A, that inhibits tyrosine kinases flk, flt-1and flt-4, tricyclodecan-9-yl xanthanante that inhibits collagen type IV synthesis (an essential scaffolding molecule for angiogenesis), and rIL-12. Interleukin 12 induces interferon gamma, which, in turn, induces protein IP-10, a known inhibitor of angiogenesis. The mechanism of action of IP-10 is not known.

These approaches have all been clinically tested in humans and have shown promise as anti-tumor agents. Any of these agents should show some activity against rete formation. Even partial inhibition should show some effects on the growth curve of the larva, if the rete is essential for its nutrient acquisition. As new classes of inhibitors are discovered and become available to the research community, a more comprehensive approach to studying the circulatory rete will no doubt evolve.

 

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