Amboceptor

Microbiological, virological, bacteriological, immunological, medical, epidemiological, historical, anecdotal

Category: Anecdotal

Medical privacy, 1950 style

Without really working in a medical field, I still hear a lot about medical privacy and HIPAA. And how any public presentation of patient information needs to be anonymized, any identifying details need to be removed, any irrelevant personal details need to be removed, populations need to be profiled rather than individuals, and generally all publications about human patients must run through a battery of administrators to make sure everything is in compliance with federal rules, state rules, institutional rules, the institutional rules of other institutions that collaborated with us, and heaven knows what else.

Not so long ago, the standards were different. Today we’ll look at a 1950 article on “Primary Cutaneous Cryptococcosis” from Archives of Dermatology and Syphilology (later renamed Archives of Dermatology, and then JAMA Dermatology). An actual doctor could explain what parts of this article would be scrubbed now, and what parts are still OK. I don’t really know. But what struck me was that nearly all the personal details seem unrelated to the actual topic of the article. Meanwhile, author William M. Gandy, M.D. points out that “family history was irrelevant”. Indeed, it would be even more irrelevant to point out the patient’s mother’s alcoholism, or grandfather’s yaws and phossy jaw.

gandy-title

Information we can glean from the piece:

– “The patient had been under continuous observation and treatment at the Charity Hospital of New Orleans since July 2, 1945. Successive complaints brought her at one time or another to the attention of the medical, surgical, gynecologic and dermatologic services.”

– “She stated that she had been ‘anemic’ and afflicted with ‘enlarged glands’ since childhood. She had measles, mumps, chickenpox and scarlet fever in early life.”

– She was “first seen by us” (the dermatology clinic) on August 14, 1947, at age 29.

– She was white and a lifetime resident of New Orleans, Louisiana.

– At this time she was divorced with one child.

– That child was conceived in 1942 in Cleveland, Ohio, but born in New Orleans.

– For months after the birth of the child, she was given weekly X-ray treatments for “extensive” genital warts. The “Roentgen rays” did no good and she was then given podophyllin ointment, which led to “a severe burn followed by a slough of much of the vulva”.

– During 1942 she was diagnosed with Boeck‘s sarcoid.

– During 1947 she had stones removed from her right kidney.

– During 1947 she underwent a vulvectomy and a ureterotomy.

– The whole point of this paper, a cutaneous Cryptococcus infection on her face, was probably the least of her problems and had little to do with all this other stuff anyway.

– Very unusual for cryptococcosis, this infection was limited to a patch of skin and not found in the blood, cerebrospinal fluid, urine, sputum or mucosal swabs.

– 12 mice were injected with half a milliliter of a broth culture of her strain of Cryptococcus. 6 of the mice died within 12 days, and the remaining 6 died within 21 days.

– Between the end of 1947 and April 1949 she left Charity Hospital, married her second husband, and returned to Charity Hospital, this time the urology clinic, after developing a renal abscess and uremia. This was not a Cryptococcus infection.

– “Further physical and laboratory investigations were not possible at the time because of the patient’s uncooperative attitude and the severity of her illness.”

– Finally… here’s a picture of her face.

gandy-cryptococcus-1950

If you know this woman, you may be glad to know that she received a large amount of medical care free from top clinicians, as Charity Hospital was adjacent to the LSU Health Sciences Center. Was this care better than what would have been provided in 1736, when the hospital was founded? Of course! What kind of question is that? Or anyway, I think so, but I’m no historian.

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Confusing movie science: S+H+E (1980)

She. A title familiar to millions, from H. Rider Haggard’s 1887 adventure novel that’s had a half-dozen film adaptations, giving actresses like Helen Gahagan and Ursula Andress a chance to be statuesque and intimidating as the titular 2,000-year-old sorceress worshiped by remote tribesmen.she-poster-1979

This is not one of those films. Having bought it along with other 1980s VHS tapes, I expected the 1982 adaptation of Haggard’s novel starring Sandahl Bergman of Conan the Barbarian and Hell Comes to Frogtown fame. Instead, it’s the acronymic S+H+E: Security Hazards Expert, which aired on CBS in February 1980. Filmed on location in “Italy and Berlin”, this was an ambitious attempted pilot for a Charlie’s Angels-esque series about a female secret agent played by Cornelia Sharpe, wife of producer Martin Bregman.

She is as effective as you could hope for as a “Diana Rigg type”. The whole movie is entertaining, despite the random elements that only make sense if there are subsequent episodes (like her Italian boyfriend who wants her to retire and settle down) and the soundtrack consisting of the same song over the opening credits, closing credits, every montage, and every action sequence. Adding to the enjoyment was the single trailer that preceded the movie, for another CBS TV movie starring Dyan Cannon as a madam who was elected mayor of Sausalito, California.

* * *

she-opening

The last thing I expected from this movie was microbiological blog fodder. But after hearing the characters talk about the science that underlies the plot, I had to stop and figure out exactly why it didn’t make sense.

* * *

First, we’re introduced to the wine scientists. The whole movie is on YouTube as of today; start around the 23-minute mark for this conversation. Charming wine magnate Cesare Magnasco (Omar Sharif) is showing our heroine Lavinia Kean around his sinister winery. Frau Doktor Biebling is played by sixties icon Anita Ekberg, in what I believe was her last English-language role.

  • Cesare: Miss Blake, this is our distinguished oenologist, Frau Doktor Biebling.
  • Lavinia: How do you do?
  • Biebling: (silence)
  • Cesare: Dr. Biebling is a genius. A Nobel Prize nominee in parasitology from the University of Heidelberg. Doctor, tell Miss Blake what we do here.
  • Biebling: We are approaching a very critical phase in our latest experiment, Barone. It requires concentration and my closest attention.
  • Cesare: Per favore. For her American readers. To make them drink more Magnasco!
  • Biebling: Our chief concern is to protect the vines. Particularly from a genus of insects of the family Phylloxera.
  • Lavinia: Which of the 32 known species do you specialize in?
  • Biebling: (silence)
  • Cesare: They destroyed millions of acres of grapes when first brought to Europe.
  • Biebling: (withering stare) From America.
  • Lavinia: (smirking) I’m terribly sorry! I won’t disturb you any longer.
  • As they leave Biebling’s lab, Magnasco points to two Petri dishes, saying “Experimental cultures”. Another scientist asks Biebling mockingly, “Why don’t you develop an anti-jealousy microbe?”

The issue here is the word “parasitology”. Although the Phylloxera family of aphids do act as parasites to grapevines, someone who studies them would be an entomologist. Parasitology generally refers to parasites of animals — especially worms or microscopic eukaryotes like malaria, Toxoplasma or Giardia. Wikipedia claims it also refers to organisms like fleas and lice, but even this only extends to parasites of animals, not plants.

It’s possible that instead of being an expert on aphids, she is an expert on microscopic parasites of aphids. This hypothesis is supported by the “experimental cultures”, which look like bacterial broth. However, I don’t think there are any such microbes, whether eukaryotic or bacterial. There are methods of aphid control using aphid parasites… but those parasites are tiny wasps. So either way, she’d be an entomologist!

Finally, calling someone a “Nobel Prize nominee in parasitology” sort of implies that there is a Nobel Prize in Parasitology, which there isn’t. And although it is prestigious to be nominated for a Nobel Prize, it’s not quite like the Academy Awards. The 2014 Nobel Prize in Physiology of Medicine had 263 nominees.

* * *

But this is mere pedantry. The really confounding exchange is near the end, around minute 73. By now, the good guys have learned that Magnasco & Co. are holding the world’s petroleum supply hostage with something called “A.P.M.” Lavinia Kean, and her assistant/minder/colleauge Lacey (basically Bosley from Charlie’s Angels), have snuck into Frau Doktor Biebling’s laboratory again. They snoop around, and when they’re discovered, Lavinia injects Biebling with some sort of sedative, injected via flying mechanical bug.

  • Biebling: You again!
  • Lavinia: Do unto others… darling.
  • Biebling: I should have put you to sleep permanently. (passes out)
  • Lacey: (points to page of notes) “Anti-Petroleum Microbe”?
  • Both together: A.P.M.!
  • (she smears the orange liquid from one of the cultures on a slide, and they look at it under a microscope. Wriggling animalcules are seen through the lens)
  • Lavinia: There it is. A simple amoeba-type microbe found in tide pools. Elsa’s a parasitologist, but she wasn’t working on Phylloxera. She was developing A.P.M.
  • Lacey: I’m lost.
  • Lavinia: She fed and refined the algae with special nutrients. Now it’s a virulent strain that devours petroleum.
  • Lacey: I’m still lost.
  • Lavinia: When A.P.M. is mixed with petroleum it multiplies like crazy. Gasoline is refined petroleum.
  • She goes on to demonstrate how gasoline is flammable, but when mixed with A.P.M. it goes all foamy and inert.

This makes it clear that Frau Doktor Biebling is an expert on microbes, not so much on aphids.

This is gasoline. This is gasoline on A.P.M.

This is gasoline. This is gasoline on A.P.M.

Her work for Magnasco has been a matter of encouraging a certain microbe to grow on different substrates, akin to the classic experiments in microbial evolution where bacteria start to thrive on some molecule that used to poison them. With “A.P.M.”, they have made a microbe which “devours petroleum”, rendering it a foamy mass of uselessness. The notion of microbes engineered to eat hydrocarbons has been invoked for decades, generally as a good thing (cleaning up oil spills). Here it’s presented as a sort of plague, which if it ever enters a pipeline will spread and spread until it consumes our entire inexorably interconnected petroleum supply.

dave-barry-oil-slicks

In a column from the 1980s, Dave Barry sees petroleum-eating microbes as neither plague nor panacea.

One problem here is that the new, oil-eating microbe is described as a “virulent strain”. That’s not what “virulent” means. A virulent strain would be one that is particularly harmful or deadly to the organism it infects. A.P.M., though scary, isn’t infecting anything — it’s just consuming certain nutrients.

And then there’s that word “parasitologist” again.

It seems that in the world of this movie, vines are protected against aphids by spraying them with an infectious agent that kills the bugs. So she could be a “parasitologist”, if the infectious agent is eukaryotic rather than bacterial.

A.P.M. under the microscope

A.P.M. under the microscope

Amoeba proteus (click for source)

Amoeba (click for source)

And indeed, it’s described as “a simple amoeba-type microbe”. Judge for yourself if that view through the microscope shows an amoeba-type microbe. What are some other possibilities? They appear filamentous, but not rigid enough to be filamentous bacteria, and I don’t see the branching that would indicate fungal hyphae. Maybe they are fragments of some larger structure. Could these be strands of filamentous algae, but photographed in a way that washes out the color?

Yes! In addition to being an amoeba, A.P.M. is described as “algae”. It normally lives in tidal pools, but has been acclimated to life in petroleum. And here I admit a misconception of my own: I thought algae would be a poor choice for evil scientists seeking speedy evolution of new abilities, compared to bacteria. But this might not be the case – Bradley Olson’s evolutionary biology lab at Kansas State, for example, uses algae as a model organism.

The question now has to be asked: Does Frau Doktor Biebling do any work at all related to grapevines? What did she promise to do in her grant proposals?

* * *

To read about a real-life salt-loving microbe that eats oil slicks, go to the MicrobeWiki entry on Alcanivorax.

To learn more about colorless algae that are also amoebae, infectious parasites of oil and studied by entomological oenologists, watch S+H+E.

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"The only way to deactivate A.P.M. is to freeze it in CO2, put it in metal containers and sink it in the Artic Ocean."

“The only way to deactivate A.P.M. is to freeze it in CO2, put it in metal containers and sink it in the Artic Ocean.” William Traylor (Lacey) and Cornelia Sharpe (Lavinia Kean) in S+H+E

Medical photography, Dorothea Lange style

There are six illustrations in this paper. The first two show the layout of the sinuses. The other four are haunting.

From M.H. Gill (1906), Diseases of the Maxillary Sinus, Yale Medical Journal XII(9):821-829:

maxillary-sinus-1

maxillary-sinus-2

maxillary-sinus-3

maxillary-sinus-4

These people (three adult men with abscesses and “a female aged ten” with cancer) got sick and came to St. Francis Hospital around 110 years ago. We hope they got better.

Pubmedwhack: Immuno-DTO

Today’s Pubmedwhack comes from the world of unstable metals and electron microscopy. For the definition of “Pubmedwhack”, see this earlier post.

* * *

Following the invention of immunofluorescence, scientists developed other methods for using labeled antibodies to identify certain proteins or substances under a microscope.

There’s immunohistochemistry (IHC), in which the antibodies are labeled not with a fluorescent marker, but with an enzyme which produces a visible reaction in the presence of a substrate. This is less precise, but lets you see your protein of choice under visible light while also looking at the structure of the tissue. The slides labeled this way last longer instead of being quenched by the microscope’s light source.

You can also use labeled antibodies to see things under the electron microscope (immuno-electron microscopy or immuno-EM). In 1960 Rifkin et al. (1) published an image of virus particles on a cell surface, labeled with ferritin-conjugated antibodies. As you can see, instead of the labeled antibodies changing the color of a region of the cell, each individual labeled antibody is visible as a “granule”.

rifkin-1960-ferritin

from Rifkin et al (1960), Nature 187:1094

This was made possible by an innovation published one year earlier, entitled Preparation of an electron-dense antibody conjugate (2). Ferritin is a small protein which just about all organisms use as an iron carrier. When “iron-loaded”, almost a quarter of its mass is iron atoms. Therefore this is an especially electron-dense molecule, visible as a dark spot under the electron microscope, as seen above. Soon, further advances let scientists see ferritin-labeled structures inside cells.

* * *

For about a decade ferritin was the label of choice for immuno-electron microscopy. Then in 1971, a new technique came along (3, 4), in which antibodies were mixed with a solution of colloidal gold until they absorbed to the metal’s surface. Gold-labeled antibodies could be separated from free antibodies by centrifugation. Immunogold is still the dominant immuno-EM staining method 40 years later.

from Faulk (1971), Nature New Biology 231:101

from Faulk et al (1971), Nature New Biology 231:101

* * *

In the 1960s, other techniques were created for immuno-EM. I have almost no EM experience and don’t know the pros and cons, but clearly there was a desire to get rid of the protein element of the electron-dense antibody label, and just attach the antibody to a metal ion. The protein was unnecessarily big, and subject to denaturation. So the 1960s also saw a lot of papers using antibodies labeled with mercury (technically the diazonium salt of tetraacetoxymercuriarsanilic acid (5) and p-(aminophenyl)-mercuric acetate (6), phrases which mean little to me).

from Zhdanov et al (1965), J Histochem Cytochem

from Zhdanov et al (1965), J Histochem Cytochem 13:684

There were also studies using antibodies labeled with heavier metals. In his long career, Ludwig Sternberger and his lab (at Johns Hopkins and elsewhere) invented several microscopy techniques, with the most important probably being the horseradish peroxidase (see original paper (7), and appreciation of it as a “citation classic”).

He also spent much of the 1960s devising improved metal-based antibody labels for electron microscopy, including immunouranium (8), immunouranium with added osmium for enhanced contrast (9), and finally immuno-diazothioether-osmium tetroxide (10), or immuno-DTO. The uranium methods seem somewhat useful, but as far as I can tell were only used by Sternberger’s own lab. Immuno-DTO in particular seemed almost unusable; they used it more than once, but a Pubmed search for “Immuno-DTO” only returns one result (11). As Sternberger himself says in a review (12) of his and other techniques:

Unfortunately, the diazotized diazothioethers are not very stable even at Dry Ice temperatures and the solid deteriorates in a few days. Therefore, it was not surprising to observe that the osmnium tetroxide-binding iower of diazothioether antibodies was unstable, even on storage in liquid nitrogen.

Oh well, it was a nice idea.

* * *

1. Rifkind RA, Hsu KC, Morgan C, Seegal BC, Knox AW, Rose HM (1960). Use of Ferritin-Conjugated Antibody to Localize Antigen by Electron Microscopy. Nature 187:1094-1095.

2. Singer SJ (1959). Preparation of an Electron-dense Antibody Conjugate. Nature 183:1523-1524.

3.Faulk WP, Taylor GM (1971). An Immunocolloid Method for the Electron Microscopy. Immunochemistry 8(11):1081-1083.

4. Faulk WP, Vyas GN, Phillips CA, Fudenberg HH, Chism K (1971). Passive Haemagglutination Test for Anti-rhinovirus Antibodies. Nature New Biology 231:101-104.

5. Pepe FA (1961). The Use of Specific Antibody in Electron Microscopy: I: Preparation of Mercury-Labeled Antibody. J Biophys Biochem Cytol 11(3):515-520.

6. Zhdanov VM, Azadova NB, Kulberg AY (1965). The Use of Antibody Labeled with an Organic Mercury Compound in Electron Microscopy. J Histochem Cytochem 13(8):694-687.

7. Sternberger LA, Hardy PH Jr, Cuculis JJ, Meyer HG (1970). The Unlabeled Antibody Enzyme Method of Immunohistochemistry: Preparation and Properties of Soluble Antigen-Antibody Complex (Horseradish Peroxidase-Antihorseradish Peroxidase) and its Use in Identification of Spirochetes. J Histochem Cytochem 18(5):315-333.

8. Donati EJ, Figge FHJ, Sternberger LA (1965). Staining of Vaccinia Antigen by Immunouranium Technique. Exp Mol Pathol 4(1):126-129.

9. Sternberger LA, Hanker JS, Donati EJ, Petrali JP, Seligman AM (1966). Method for Enhancement of Electron Microscopic Visualization of Embedded Antigen by Bridging Osmium to Uranium Antibody with Thiocarbohydrazide. J Histochem Cytochem 14(10):711-718.

10. Donati EJ, Petrali JP, Sternberger LA (1966). Formation of Vaccinia Antigen Studied by Immunouranium and Immuno-diaxothioether-osmium tetroxide Techniques. Exp Mol Pathol Apr:Suppl 3:59-74.

11. Sternberger LA, Donati EJ, Hanker JS, Seligman AM (1966). Immuno-diazothioether-osmium tetroxide (immuno-DTO) technique for staining embedded antigen in electron microscopy. Exp Mol Pathol Apr:Suppl 3:36-43.

12. Sternberger LA (1967). Electron Microscopic Immunocytochemistry: A Review. J Histochem Cytochem 15(3):139-159.