Comparative Medicine
Volume 56, Number 2 (April 2006)
Phenotypic of aquaporin mutants in genetically altered mice (pages 96-104)
Summary
Aquaporins are water-channel proteins found in all plants in animals. Fluid movement through aquaporins is passive and nondirectional. The 'gold standard' for characterizing aquaporin function is the Xenopus oocyte swelling assay where aquaporin RNA is injected into frog eggs and the ce'lls transcription and translation machinery convert it to protein and insert it in the oocyte membrane. Swelling of these oocytes when transferred from iso-to hypotonic buffer confirms that functional water channels were inserted. Uptake of radiolabeled compounds and changes in membrane potential can also be measured using the frog oocyte swelling assay. Aquaporins are divided into 2 groups: 1) those that are water permeable 2) those that are permeable to water and glycerol, urea, and other small molecules. There are a number of aquaporin knockout mouse models, many of which were created using 129S5 or CD-1 embryonic stem cells and 129S5 or CD-1 blastocysts and breeding founder mice with B6 mice CD-1 mice.The author believes that several of the phenotypes observed in these knockouts (e.g. cataracts by 6 weeks of age in the Cat-Fr AQP0 K/0 mouse, PU/PD and renal lesions in AQP1, 2 and 3 K/O mice, seizure resistance and impaired hearing of AQP4 mice, and obesity in AQP7 K/O mice) may be partially due to the effects of background strain as opposed to being caused solely by the respective aquaporin null mutations.
Questions:
Question 1: Name the “gold standard” for characterizing aquaporin function.
Question 2: Which aquaporin is found exclusively in terminally differentiated lens fiber cells where it
comprises >50% of the total membrane protein.
Question 3: What is the effect of defects in AQP0 in humans andmice?
Question 4: Which aquaporin knockout mouse develops polycystic kidneys and dies at weaning due to renal
failure?
Question 5: Which aquaporin knockout mouse has been used to evaluate the role of aquaporins in skin
physiology? What skin characteristics were observed in this mouse?
Question 6: Abnormalities in which aquaporin’s distribution and trafficking is thought to be involved
in the Sjögren’s syndrome phenotype in humans? What is the Sjögren’s syndrome in humans?
Answers:
Answer 1: The Xenopus oocyte swelling assay where aquaporin RNA is injected into frog eggs, where it is transcribed and translated into protein and inserted into the oocyte membrane. The presence of functional water channels is determined by moving oocytes from iso- hypotonic buffer solutions and measuring oocyte swelling and rupture.
Answer 2: AQP0.
Answer 3: At least 2 spontaneous mutant mouse models of cataract formation have been associated with AQP0: Cataract Fraser (Cat-Fr) mouse which has a lack of adequate AQP0 production and cataract formation by 6 weeks of age, Lop mouse with Lop10 mutation causing protein trafficking defects of AQP0 and which is microphthalmic with cataract formation at birth
Answer 4: AQP 11 , the newest aquaporin for which a knockout or knock-in mouse model is published (as of April 2006)
Answer 5: AQP3 knock-out mouse which has impaired hydration and decreased glycerol content of the skin’s stratum corneum.
Answer 6: AQP5 found in the apical membranes of exocrine glands, including salivary, lacrimal, sweat, pulmonary submucosal glands, and Type I pneumocytes. Sjögren’s syndrome: xerostoma (drymouth) and keratoconjunctivitis sicca due to inadequate saliva and tear production.
Feasibility and limits of an orthotopic human colon cancer model in nude mice (pages 105-109)
Study has been conducted to develop an accurate colorectal cancer model in nude mice, with three factors; stable local growth, tumor cell dissemination in the bone marrow, and reproducible metastatic capacity. They orthotopically transplanted histologically intact human colorectal cancer tissue into nude mice. Then the successful local tumor growth, tumor tissues were retransplanted as many as 9 times in serial passage. Stable local tumor growth rates were detected in 73.4% of all mice. The lymph node and hepatic metastasis rates were low, at 18.4% and 4.9% respectively. Lung metastases were not detected in any of the animals.
Authors suggest that the observed induction of minimal residual disease after orthotopic implantation of the intact human colon cancer in animals with lymph node and hepatic metastasis might be improved if established colon cancer cell lines were used.
Questions:
1-Which one is the characteristic of the minimal residual disease (MRD):
a)is characterized by disseminated tumor cell in blood
b)is characterized by disseminated tumor cell in bone marrow
c)is characterized by disseminated tumor cell in lymph nodes
d)is a negative prognostic marker in solid tumors
e)All above
2-Which component of simple epithelium-derived cells is a sensitive and specific detector of disseminated epithelium-derived cells against the mesanchymal background of the bone marrow?
3-A commonly used method to detect Cytokeratin 20?
4-Is the CK20 positivity of the mice with lymph node and hepatic metastases, necessarily a consequence of MRD in the course of metastatic disease?
Answers
1-e
2-Cytokeratin 20(CK20), RT-PCR
3- CK20 RT-PCR
4-No
Occurrence of Helicobacter species other than H. hepaticus in laboratory mice and rats in Sweden (pages 110-113)
This study analyzed 63 mouse and 42 rat samples from samples that were previously positive for Helicobacter spp. but negative for H. hepaticus in the National Veterinary Institute in Sweden . The samples were analyzed via partial 16S rDNA sequence analysis which allowed the detection of both known and unknown Helicobacter spp. The purpose was to determine which Helicobacter spp. besides H. hepaticus was present in Sweden ’s laboratory animal facilities. In mice the Helicobacter species found were, H. ganmani, H. typhlonius, H. rodentium. They also found an uncharacterized strain that was similar to the Helicobacter spp. hamster B and a possibly novel strain that most closely related to H. apodemus strain YMRC 000216. In rats H. ganmani (previously not reported in rats) and H. rodentium were found.
QUESTIONS:
1. Name the two Helicobacter species that was found in rats.
2. Which of these two species was previously not found in rats?
3. What was the advantage of using 16S rDNA sequence analysis to analyze these samples?
4. Helicobacter is a gram _____ bacterium
ANSWERS:
1. H. ganmani and H. rodentium
2. H. ganmani
3. It allowed detection of both known and unknown Helicobacter species
4. negative
Are repeated doses of buprenorphine detrimental to postoperative recovery after laparotomy in rats? (pages 114-118)
SUMMARY: The effects of peri- and postoperative buprenorphine administration (0.05 mg/kg, SC) on the postoperative stress response were evaluated in female Wistar rats undergoing laparotomy and bile duct isolation. Body weight, daily food and water consumption, and daily fecal and urinary outputs were monitored 7 days following surgery in buprenorphine and saline treated rats. Buprenorphine, irrespective of when treatment was initiated relative to surgery (pre-, intra-, postoperatively), had no significant effects on daily food and water consumption or daily fecal and urinary outputs compared to saline-treated animals. However, buprenorphine administration delayed the animal’s return to preoperative body weight levels following surgery. Daily buprenorphine administration for 7 days in surgically naïve rats had no effects on any of the outcomes evaluated compared with surgically naïve saline-treated rats.
QUESTIONS:
1. Buprenorphine has partial agonist activity at which of the following receptors?
a. Sigma
b. Mu
c. Kappa
d. Delta
2. The postoperative stress response has been described as having 4 phases. Match the following descriptions to the correct postoperative stress response phase (Phases 1-4).
i. Weight gain begins as the animals moves into positive nitrogen balance
ii. Immediate activation of the sympathetic-adrenomedullary and hypothalamic-pituitary-adrenocortical systems occurs
iii. Weight gain occurs due to fat accumulation
iv. Sympathetic hyperactivity ceases and hypothalamic-pituitary-adrenocortical system activity begins to wane
3. In this study, the authors found that perioperative buprenorphine administration had which of the following effects in postoperative rats compared to saline-treated rats.
a. Delayed return to pre-operative body weight levels
b. Altered daily water intake
c. Attenuated daily urinary output
d. Increased daily food consumption and fecal output
e. Induced pica
ANSWERS:
1. b. Mu
2. i. Phase 3; ii. Phase 1; iii. Phase 4; iv. Phase 2
3. a. Delayed return to pre-operative body weight levels
Detection of systemic amyloidosis in the pig-tailed macaque (Macaca nemestrina) (pages 119-127)
Amyloid is a protein which, when folded incorrectly, aggregates extracellularly.Primary amyloidosis is due to overproduction of the immunoglobulin light chain and may be neoplastic or genetic in origin. Secondary, or reactive amyloidosos is due to an accumulation of serum amyloid- an acute phase protein associated with inflammation.Amyloidosis in NHP has been associated with rheumatoid arthritis, viral infection, parasitism, chronic catheterization and enterocolitis.Amyloidosis secondary to enterocolitis, historically attributed to Shigella sp.v has been described in multiple macaque species.There is no reliable diagnostic assay, preventive measure or treatment for secondary amyloidosis. Histologic examination is the gold standard for diagnosis of amyloid.The amyloid fibers bind Congo Red and exhibit green birefringence with polarization. The purpose of this study was to evaluate 12 diagnostic tests for detection of subclinical amyloidosis.
For12 months, animals that were selected for removal from the colony at the Washington National Primate Research Center were sampled and imaged. The diagnostic tests included endoscopic examination and biosy of the stomach and colon, ultrasonography of the liver, hepatic radiology, serum amyloid A(SAA)ELISA, endothelin 1 LISA, alpha-fetoprotein ELISA, tissue histology and an expanded chemistry panel including C-reactive protein, erythrocyte sedimentation rate, albumin, blood urea nitrogen, bilirubin, total cholesterol, total protein, gamma-glutyltransferase(GGT), alanine aminotransferase(ALT), aspartate aminotransferase and alkaline phosphatase(ALP).
30 female M. nemestrina monkeys were sampled.14 animals were confirmed positive for amyloid deposition based on histology. Within the positive animal population, the following tissues affectedwere:
liver 30%
GI30%
adrenals38%
kidneys 25%
spleen24%
Age was found to be a significant indicator of amyloid as affected animals had a mean age of 13.2 compared to 9.3 for unaffected animals.Hepatic radiology was predictive of amyloidosis, but ultrasonography was nondiagnostic for hepatic, adrenal, splenic and renal amyloidosis. Endoscopy did not demontrate visual GI tract lesions, however tissues from amyloid-positive animals were subjectively more friable. This study did not demonstrate an association between systemic or GI amyloidosis and parasitismor amyloidosis and viral infection.Significant differences in GGT, total cholesterol, albumin, total protein, ALP and SAA were noticed in amyloidotic animals. Albumin and total protein were decreased while the otherlevels were elevated.None of the other serum chemistries differed significantly between amyloid-positive and -negative animals.
The authors note that amyloidosis remains an antemortem diagnosis by exclusion.Although the histologic presence of amyloid allows for a positive diagnosis, the absence of amyloid is not a negative diagnosis because of sample size bias and nonuniform tissue distribution.Serology may assist in the diagnosis.Those with elevated GGT, ALP and cholesterol in combination with normal bilirubin and decreased albumin and total protein should be further evaluated.This may be considered an amyloidotic profile. Abdominal radiographs are diagnostic for hepatomegaly which in the face of an amyloidotic serologic profile, may indicate amyloidosis.In this survey, an elevated SAA with an amyloidotic serologic profile, normal clinical exam, negative viral status and radiographic hepatomegaly was diagnostic for amyloidosis.
Questions:
1.Name the 2 forms of amyloidosis and their causes.
2.The gold standard for diagnosis is histology.Name the specific diagnostic stain and tissue behavior.
3. T/F:There is no age indicator for amyloidosis.
4.T/F:There are no imaging modalities which assist in predicting amyloidosis.
5.Name theserologic changes associated with an amyloidotic profile.
Answers:
1.primary - overproduction of the immunoglobulin light chain secondary (reactive) - accumulation of serum amyloid A
2.Amyloid fibers bind Congo Red and exhibit green birefringence with polarization.
3.F.The animals with amyloidosis were significantly older.
4.F.Hepatic radiology revealing hepatomegaly was a positive predictor for hepatic amyloidosis.
5.Decreased total protein and albumin, increased GGT, ALP and cholesterol
Cortisol metabolism in the Bolivian squirrel monkey (Saimiri boliviensis boliviensis) (pages 128-135)
Summary: Compared with humans, the new world squirrel monkey has very high levels of circulating cortisol. (Human range is 5-25 µg/dl and squirrel monkey ave. was 188 µg/dl in Feb/Mar and 96 µg/dl in Aug-Oct.) Even with these high cortisol levels, the monkey maintains normal serum electrolyte values and normal blood pressure. This article examined 3 possible adaptive mechanisms:
(1) Measure circulating levels of aldosterone. Levels were similar as reported for humans.
(2) Compare activity of squirrel monkey mineralocortical receptor to human in response to cortisol and aldosterone. No difference was found.
(3) Identify predominant pathways of metabolism.
a. Found increased conversion of cortisol to cortisone (an inactive metabolite). The ratio of cortisol/cortisone was 0.93 for the monkey vs. 3.0-7.3 for humans.
b. Increased urinary excretion of cortisol. High levels of both cortisol and cortisone were found in urine.
c. Found higher levels of the metabolite 6β-hydroxycortisol in urine (19% vs. 1% for humans).
Questions:
1: T/F In the squirrel monkey, a seasonal variation of cortisol level was seen.
2: Increased cortisol levels can lead to increased blood pressure by urinary retention of what electrolyte?______. Name the hormone that regulates this.______
3: Elevated serum cortisol, called ______disease, can be due to excess adrenocorticotropin secretion or an adrenal tumor.
Answers:
1: T
2: Sodium; aldosterone
3: Cushing’s
Ovine intestinal adenocarcinomas: Histologic and phenotypic comparison with human colon cancer (pages 136-?)
Summary: The authors have collected intestinal tracts from 50slaughtered sheep with visible intestinal neoplasia. They examinedthe phenotypic and histologic characteristics of tumors, andclassified them according to WHO guidelines (TNM system). Extensionto lymph nodes, peritoine and other organs was also examined for someanimals.
Histologic features of sheep intestinal neoplams appeared to be quitesimilar to human ones. All tumors were adenocarcinomas, with invasionof submucosa, muscularis layer and serosa. Metastasis was frequent,usually on peritoine (2 hepatic metastasis were observed over 22examinations). Ulcerations, fibrosis and necrosis were frequent. Thegrading of neoplasms was done according to the % of neoplastic cellsforming glands and to the vacuolisation of neoplastic cells: bothtype of tumors were equally found, as in humans (moderately or poorlydifferentiated).
Differences, compared to human bibliographic data, were the smallintestine location (all sheep neoplasia were found in jejunum), thefewer polypoid form, and the higher severity (lymph nodes involvmentand metastases).
The authors discuss the interest of sheep spontaneous intestinaladenocarcinomas, compared to rodents, as possible models of humanadvanded colonic tumors. The big size of sheep, and their overallgood condition even when tumor bearing, could support surgicaltechniques; yet a screening test to identify affected animals has tobe developped. Additionnally, it is interesting to note that theprevalence of such tumors is particularly high in New Zealand's sheep.
Questions:
1. What defines an adenocarcinoma?
2. What criteria are used to classify human colonic neoplasms?
3. The authors assume "Neoplastic cell populations had 2 to 5 mitosesper 400×field". What do you think of this number?
4. True of False: the extension of intestinal neoplasms usuallyfollows mesenteric lymphatics
5. What may explain the higher severity observed in sheep?
6. What are the major drawbacks of rodents models for human colonicadenocarcinoma?
a) rodents little size (surgical inconvenience)
b) immunologic concerns (rodents models in oncology useimmunodeficient animals)
c) rare intestinal adenocarcinomas in rodents (either spontaneous orinduced)
d) rare occurence of metastases in intestinal tumors of rodents(early endpoint due to intestinal disease)
e) all of the above
7. What screening test could be use to identify intestinal tumorbearing sheep before slaughterhouse?
8. True of False: the high prevalence of intestinal adenocarcinomasin sheep relies on genetic factors, as suggested by the heterogeneousdistribution among farms and countries
Answers:
1. Adenocarcinoma is a form of carcinoma that originates in glandulartissue. It is malignant by definition: carcinomas invade surroundingtissues and organs, and may spread to lymph nodes and distal sites(metastasis).
2. The TNM system is recommended by the World Health Organisation: T1 to 4 (depth of invasion of neoplastic cells in the intestinalwall), N 0 to 2 (0, 1 to 3 or ≥4 lymph nodes inflitrated withneoplastic cells), M 0 to 1 (no or presence of distant metastases).
3. Mitotic rate of tumors may be used for prognosis, among othercriteria. Mitotic rates <2 per 400xfield are usually reported inhuman colon adenocarcinomas (J. Clin. Pathol. 1995)
4. True
5. Examination were made on old slaughtered animals, without data ontheir health status: the invasion and metastasis may result from alack of diagnostic, compared to data collected at an earlierdiagnosis time in human.
6. a, c and d (b is not true: immunocompetent and immunodeficientrodents models are used equally in oncology; furthermore, thehistocompatibility of inbred rodents is a great advantage, thatenables grafting of tumoral or immune cells as well).
7. A screening test dosing carcinoembryonic antigen (CEA) in serumwould be a very simple option, but yet to be developped; fecal bloodtesting and colonoscopy can't be used since sheep tumors are in thejejunum; laparoscopic or ultrasound screening could be used in flockspresenting high incidence.
8. False: no such conclusion can be drawn only from the distribution(a viral factor or a polluant factor may also have an heterogenousimpact).