Enzyme-linked immunosorbent assay detection of melamine in infant formula and wheat food products.

The adulteration of food products with melamine to inflate the nitrogen content necessitates the establishment of analytical methods that can distinguish between proteinaceous ingredients and such adulterants. The specificity and ability to detect melamine by two commercial enzyme-linked immunosorbent assay (ELISA) kits were evaluated along with three protocols for sample preparation. Both ELISAs displayed cross-reactivity with ammeline, but neither was able to detect ammelide or cyanuric acid, indicating either a requirement for the 4,6-diamino-1,3,5-triazine structure or inability to bind 1,3,5-triazine-4,6-diones. The limits of detection for melamine in powder infant formula ranged from 0.2 to 3 microg/g depending on the ELISA kit and the method used to prepare the sample. The limits of detection for melamine in liquid infant formula and wheat products were <1 microg/ml and <2.5 microg/g, respectively. The ELISA kits provide an effective alternative for the analysis of samples suspected of containing melamine without relying on extensive sample preparation or expensive instrumentation.

The Large Crater Origin of SNC Meteorites.

A large body of evidence strongly suggests that the shergottite, nakhlite, and Chassigny (SNC) meteorites are from Mars. Various mechanisms for the ejection of large rocks at martian escape velocity (5 kilometers per second) have been investigated, but none has proved wholly satisfactory. This article examines a number of possible ejection and cosmic-ray exposure histories to determine which is most plausible. For each possible history, the Melosh spallation model is used to estimate the size of the crater required to produce ejecta fragments of the required size with velocities >/=5 kilometers per second and to produce a total mass of solid ejecta consistent with the observed mass flux of SNC meteorites. Estimates of crater production rates on Mars are then used to evaluate the probability that sufficiently large craters have formed during the available time. The results indicate that the SNC meteorites were probably ejected from a very large crater (> 100 kilometers in diameter) about 200 million years ago, and that cosmic-ray exposure of the recovered meteorites was initiated after collisional fragmentation of the original ejecta in space at much later times (0.5 to 10 million years ago).

VapC proteins from Mycobacterium tuberculosis share ribonuclease sequence specificity but differ in regulation and toxicity.

The chromosome of Mycobacterium tuberculosis (Mtb) contains a large number of Type II toxin-antitoxin (TA) systems. The majority of these belong to the VapBC TA family, characterised by the VapC protein consisting of a PIN domain with four conserved acidic residues, and proposed ribonuclease activity. Characterisation of five VapC (VapC1, 19, 27, 29 and 39) proteins from various regions of the Mtb chromosome using a combination of pentaprobe RNA sequences and mass spectrometry revealed a shared ribonuclease sequence-specificity with a preference for UAGG sequences. The TA complex VapBC29 is auto-regulatory and interacts with inverted repeat sequences in the vapBC29 promoter, whereas complexes VapBC1 and VapBC27 display no auto-regulatory properties. The difference in regulation could be due to the different properties of the VapB proteins, all of which belong to different VapB protein families. Regulation of the vapBC29 operon is specific, no cross-talk among Type II TA systems was observed. VapC29 is bacteriostatic when expressed in Mycobacterium smegmatis, whereas VapC1 and VapC27 displayed no toxicity upon expression in M. smegmatis. The shared sequence specificity of the five VapC proteins characterised is intriguing, we propose that the differences observed in regulation and toxicity is the key to understanding the role of these TA systems in the growth and persistence of Mtb.

Evaluation of the first pharmacist-administered vaccinations in Western Australia: a mixed-methods study.

OBJECTIVES: This study evaluated the uptake of Western Australian (WA) pharmacist vaccination services, the profiles of consumers being vaccinated and the facilitators and challenges experienced by pharmacy staff in the preparation, implementation and delivery of services. DESIGN: Mixed-methods methodology with both quantitative and qualitative data through surveys, pharmacy computer records and immuniser pharmacist interviews. SETTING: Community pharmacies in WA that provided pharmacist vaccination services between March and October 2015. PARTICIPANTS: Immuniser pharmacists from 86 pharmacies completed baseline surveys and 78 completed exit surveys; computer records from 57 pharmacies; 25 immuniser pharmacists were interviewed. MAIN OUTCOME MEASURES: Pharmacy and immuniser pharmacist profiles; pharmacist vaccination services provided and consumer profiles who accessed services. RESULTS: 15 621 influenza vaccinations were administered by immuniser pharmacists at 76 WA community pharmacies between March and October 2015. There were no major adverse events, and <1% of consumers experienced minor events which were appropriately managed. Between 12% and 17% of consumers were eligible to receive free influenza vaccinations under the National Immunisation Program but chose to have it at a pharmacy. A high percentage of vaccinations was delivered in rural and regional areas indicating that provision of pharmacist vaccination services facilitated access for rural and remote consumers. Immuniser pharmacists reported feeling confident in providing vaccination services and were of the opinion that services should be expanded to other vaccinations. Pharmacists also reported significant professional satisfaction in providing the service. All participating pharmacies intended to continue providing influenza vaccinations in 2016. CONCLUSIONS: This initial evaluation of WA pharmacist vaccination services showed that vaccine delivery was safe. Convenience and accessibility were important aspects in usage of services. There is scope to expand pharmacist vaccination services to other vaccines and younger children; however, government funding to pharmacists needs to be considered.

Performance Tested Method multiple laboratory validation study of ELISA-based assays for the detection of peanuts in food.

Performance Tested Method multiple laboratory validations for the detection of peanut protein in 4 different food matrixes were conducted under the auspices of the AOAC Research Institute. In this blind study, 3 commercially available ELISA test kits were validated: Neogen Veratox for Peanut, R-Biopharm RIDASCREEN FAST Peanut, and Tepnel BioKits for Peanut Assay. The food matrixes used were breakfast cereal, cookies, ice cream, and milk chocolate spiked at 0 and 5 ppm peanut. Analyses of the samples were conducted by laboratories representing industry and international and U.S governmental agencies. All 3 commercial test kits successfully identified spiked and peanut-free samples. The validation study required 60 analyses on test samples at the target level 5 microg peanut/g food and 60 analyses at a peanut-free level, which was designed to ensure that the lower 95% confidence limit for the sensitivity and specificity would not be <90%. The probability that a test sample contains an allergen given a prevalence rate of 5% and a positive test result using a single test kit analysis with 95% sensitivity and 95% specificity, which was demonstrated for these test kits, would be 50%. When 2 test kits are run simultaneously on all samples, the probability becomes 95%. It is therefore recommended that all field samples be analyzed with at least 2 of the validated kits.

Ras oncogene mutations in benign and malignant thyroid neoplasms.

Current models for tumorigenesis propose that a series of genetic alterations occur during the progression from the normal cell to the malignant phenotype. Mutations in each of the three ras genes (K-ras, H-ras, and N-ras) have been identified in many human neoplasms, including thyroid cancer. In this study we examined genomic DNA from benign and malignant thyroid neoplasms for mutations that are known to activate the ras oncogenes (codons 12, 13, and 61). DNA from frozen surgically excised tissue (n = 8) and from formalin-fixed paraffin-embedded tissue (n = 30) was amplified by the polymerase chain reaction and screened for mutations using oligonucleotide-specific hybridization. No mutations were identified in follicular adenomas (n = 9). In follicular carcinomas, 2 of 14 tumors contained mutations (N-ras 61, Gln to Arg), and both of these patients had bone metastases. One of 15 papillary carcinomas had a ras mutation (H-ras 12, Gly to Ser). In contrast to other studies, we found that ras mutations are relatively uncommon in both benign and malignant thyroid neoplasms. Studies of larger numbers of tumors and comparisons of different patient populations will be required to assess a possible association of mutations in N-ras 61 with clinically aggressive follicular cancer.

Congenital goiter and the development of metastatic follicular carcinoma with evidence for a leak of nonhormonal iodide: clinical, pathological, kinetic, and biochemical studies and a review of the literature.

We report a large kindred of patients with congenital goiter, followed for 15 yr, in which two siblings (one male and one female) developed metastatic follicular thyroid carcinoma. These two patients were evaluated by iodine kinetic analysis. None of the classical defects of T4 biosynthesis was present in either patient. Rather, both patients had extremely rapid rates of iodine turnover, with elevated 131I uptake and excessive spillage of iodide in the urine. Serum iodoalbumin was present, probably as a nonspecific result of glandular hyperplasia. Iodine kinetic analysis after the ingestion of potassium perchlorate and methimazole was compatible with a leak of nonhormonal iodide from the thyroid. It is not possible to determine whether this iodide leak is the primary pathogenetic defect or is secondary to another unidentified abnormality. The unprecedented development of metastatic thyroid cancer in patients with congenital goiter occurred, in both instances years after subtotal thyroidectomy without thyroid hormone replacement therapy, suggesting a role for TSH in the genesis of human thyroid cancer. On the basis of our study of these patients and a review of the literature, we conclude that TSH is likely to be a factor in the induction of human follicular thyroid carcinoma.

Identification of a novel transglutaminase from the filarial parasite Brugia malayi and its role in growth and development.

Recently, we reported the presence of a putative transglutaminase in adult female worms of Brugia malayi [1]. The enzyme activity was shown to be essential for in utero growth and development of microfilariae. Here, we demonstrate that adult worms of B. malayi have a large amount of epsilon-(gamma-glutamyl)lysine isopeptide bonds, a product of physiologically active transglutaminase. A 25-kDa immunoreactive band detected in female worm extracts by a monospecific monoclonal antibody (CUB 7401) against guinea pig liver transglutaminase was associated with the enzymatic activity. Unlike the mammalian enzyme, the parasite enzyme did not require Ca2+ for its catalytic activity. Furthermore, in utero developing embryos, especially during early stages of development, contained very high amounts of this enzyme. Adult female worms contained several proteins that could serve as suitable substrates for the enzyme. Inhibition of the enzyme activity by an enzyme-specific pseudosubstrate, monodansylcadaverine, led to a time- and dose-dependent inhibition of microfilariae production and release by gravid female worms. The inhibition of microfilariae production was due to the inhibition of transglutaminase-catalyzed crosslinking of parasite proteins that in turn seemed to be essential for in utero growth and development of the embryos. The results suggest that transglutaminase-catalyzed reactions may play an important role during early development of embryos to mature microfilariae inside the adult female worms of filarial parasites.

Thyroid papillary carcinoma. Pathological and philosophical controversies.

An encapsulated thyroid tumor with a papillary architecture is statistically much more likely to be a hyperplastic follicular adenoma than a papillary carcinoma, for the latter are uncommonly encapsulated. Hyperfunction of such a tumor is diagnostic of an adenoma. Various histopathologic features are useful in the differential diagnosis, but these are more accurately termed guidelines than criteria. The presence of psammoma bodies, fibrovascular stalks of papillae and pale nuclear changes are probably the most reliable histopathologic features of a carcinoma. The value of needle biopsy as a diagnostic tool for the screening of thyroid nodules is emphasized. Encapsulated variants of papillary carcinoma are discussed, including the rare pure follicular variant and those intermixed with adenomatous components, suggesting the possibility that some papillary carcinomas might arise in a preexisting adenoma. When the histopathologic diagnosis is equivocal, a benign interpretation is favored since encapsulated papillary carcinomas are very low grade, have an unusually favorable prognosis, and need no further resection. The polarization in past years of clinicians into radical and conservative operative fractions is reviewed. This stemmed from different interpretations of the presence of lymph node metastases in a majority of cases and the high frequency of intraglandular microscopic dissemination of neoplasm. It is clear that biological behavior of this unusual cancer is more important than a literal interpretation of the pathologic findings as a determinant for patient management. The surgical pathologist should be mindful that he plays an important role in patient management, not only by providing a diagnosis but also by serving as a consultant to the surgeon in pathologic correlation.

Multiple branchial cleft-like cysts in Hashimoto's thyroiditis.

We report two cases of multiple intrathyroidal cysts in women with Hashimoto's thyroiditis. The cysts were lined by squamous and focally columnar epithelium, and they were surrounded by follicular lymphoid tissue and a fibrous capsule. They appeared similar to branchial cleft cysts of the lateral neck and were distinct from previously reported types of thyroid cysts. Although the histogenesis of these lesions is unclear, they are probably derived from developmental rests. A relationship between the cystic enlargement of these rests and the Hashimoto's disease seems likely.

Pathologic features of prognostic significance in adrenocortical carcinoma.

There are currently no well-established pathologic prognostic factors helpful in distinguishing low versus high grade adrenocortical carcinomas. The effect of 11 pathologic parameters on survival was investigated in 42 cases of adrenocortical carcinoma. Only one variable, mitotic rate, had a strong statistical association with patient outcome. The 21 patients with carcinomas with greater than 20 mitoses per 50 high power fields (hpf) had a median survival of 14 months, whereas the 21 patients with carcinomas with less than or equal to 20 mitoses had a median survival of 58 months (p less than 0.02). The presence of atypical mitoses, capsular invasion, tumor weight greater than 250 g, and size greater than 10 cm each showed a marginal statistical association with poor survival (p less than 0.06), whereas other features assessed, such as nuclear grade, presence of necrosis or of venous or sinusoidal invasion, character of the tumor cell cytoplasm, or architectural pattern, showed no statistical significance in predicting survival. It is proposed that adrenal cortical carcinomas with greater than 20 mitoses be designated high grade, whereas tumors with less than or equal to 20 mitoses be designated low grade.

Functioning oxyphil cell adenomas of the parathyroid gland. A study of 15 cases.

Parathyroid adenomas composed predominantly of chief cells are the most frequent cause of primary hyperparathyroidism. Until as recently as 1978, the rare oxyphil cell parathyroid adenoma was generally considered nonfunctioning. A retrospective review of 500 consecutive patients at the Massachusetts General Hospital with a diagnosis of hyperparathyroidism associated with parathyroid adenoma during the years 1979-1987 yielded 15 (3.0%) oxyphil cell adenomas. A total of 65 case reports of hyperparathyroidism associated with a diagnosis of oxyphil cell adenomas were reviewed, applying the same diagnostic criteria used in case selection for the present series. These criteria include: (a) at least 90% composition of the adenoma by oxyphil cells; (b) biopsy or excision of a second histologically normal parathyroid gland to help rule out hyperplasia; and (c) postoperative alleviation of hypercalcemia. More than 50% of the previously reported cases did not conform to these criteria. The findings in the present study further document the entity of hyperparathyroidism caused by oxyphil cell parathyroid adenomas and suggest criteria guidelines for this rare diagnosis.

Papillary carcinoma of the thyroid, oxyphil cell type, "clear cell" variant: a light- and electron-microscopic study.

A unique case of papillary carcinoma of the thyroid having the combined features of three morphological subtypes--tall cell, clear cell and Hürthle cell--has been studied by light and electron microscopy. The distinctive neoplastic cells had an oxyphilic basal zone, a mid-placed nucleus and a clear apical region. Ultrastructurally, the cytoplasm was virtually filled with mitochondria, characteristic of Hürthle cells, but the unusual finding was the marked distention and emptiness of those mitochondria located in the apical zone, accounting for the clear phenomenon noted by light microscopy. In contrast, the mitochondria in the basal, or oxyphilic, part of the cells were intact. While a few cases of clear cell and papillary clear-cell carcinoma of the thyroid have been studied by electron microscopy previously, the clear change has never been attributed to dilated mitochondria, but rather to the presence of glycogen. The reason for the mitochondrial swelling is not answered, but it is probably an in vivo effect. Some of the other characteristics of papillary carcinoma of the thyroid described previously, such as microvilli, absence of colloid, infolded nuclei and nuclear bodies, were also present in this case. However, ground-glass nuclei, a frequently reported feature, were not found.

A direct fluorescence technique for the rapid detection of sheathed microfilariae in blood smears.

Thick and thin blood smears containing microfilariae of Wuchereria bancrofti, Loa loa, Brugia malayi, Brugia pahangi, Brugia patei or Acanthocheilonema viteae were prepared from either cryopreserved blood samples or from freshly collected blood, fixed in methanol and treated with a fluoresceinated lectin wheat germ agglutinin. Sheathed microfilariae of W. bancrofti, L. loa, B. malayi, B. pahangi and B. patei in the blood smears could be easily detected and counted using a fluorescence assay. The unsheathed microfilaria of Acanthocheilonema viteae did not fluoresce. The possibility of adapting this technique, which does not require the use of parasite specific antibody for the sensitive, parasitological detection of filarial infections, is discussed.

Lectin binding to larval stages of Brugia patei.

Surface properties of microfilariae (mf) and infective larvae of Brugia patei were investigated to compare them to previous studies with the other brugian species. Of all the lectins tested, only wheat germ agglutinin (WGA) binds to the sheath surface of mf indicating the presence of N-acetyl-D-glucosamine as a major surface carbohydrate. However, cuticle of infective larvae failed to show binding of these lectins. Enzyme treatment of mf with N-acetyl-D-glucosaminidase and L-fucosidase has exposed D-mannose, N-acetyl-D-galactosamine and L-fucose on the sheath surface. The binding of lectins to intact mf and to enzyme-treated mf appeared to be specific as pretreatment with specific inhibitory sugars completely abolished the binding activity. This is the first study conducted with this filarial parasite and it established that B. patei is similar to other species of Brugia but differs from Wuchereria in its surface-lectin binding properties.

Regulatory cytokines in the lymphatic pathology of athymic mice infected with Brugia malayi.

To investigate whether Brugia malayi-induced lymphatic inflammation is due to production of pro-inflammatory cytokines, we determined the lymph and serum levels of interleukin (IL)-1, IL-6, tumor necrosis factor (TNF)-alpha and granulocyte-macrophage colony stimulating factor (GM-CSF) using enzyme immunoassays. Serum from normal and infected mice did not show elevated cytokine concentrations. Samples of lymph from parasitized lymphatics had significantly increased levels of IL-1 (range = 6-1620 pg/ml), IL-6 (19-17,800 pg/ml), TNF-alpha (19-2000 pg/ml) and GM-CSF (4-275 pg/ml). The anti-inflammatory cytokine IL-4 (7-12 pg/ml) was in the normal range and no increase in interferon (INF)-gamma was detected in lymph samples. The data suggest that increased levels of mediators or cytokines localized in the lymphatics may be important contributors to massive lymphatic dilation and inflammation.

A comparative study of flow cytometry and histopathologic findings in thyroid follicular carcinomas and adenomas.

As a possible diagnostic aid in the often difficult histopathologic distinction of thyroid follicular carcinomas from adenomas based on invasion most flow cytometry studies have indicated a higher aneuploidy incidence in carcinomas. However, these reports often are difficult to analyze mainly due to nonuniformity of pathologic diagnostic criteria. The present study compares the flow cytometry results of 65 follicular tumors with pathologic findings based on the World Health Organization's specific diagnostic and staging criteria. Aneuploidy was significantly higher in the 28 cancers than in the 27 hypercellular (fetal and embryonal) adenomas (57% v 22%; P = .02). There was a high percentage of aneuploidy (75%; nine of 12 cases) in the widely invasive follicular carcinomas, compared with 40% (six of 15 cases) in the minimally invasive carcinomas, 22% (six of 27 cases) in the hypercellular adenomas, and 10% (one of 10 cases) in the normofollicular or macrofollicular adenomas. However, aneuploidy was not significantly different between the most difficult differential histopathologic diagnoses of minimally invasive follicular carcinoma (40%; six of 15 cases) and hypercellular adenoma (22%; six of 27 cases) (P = .12). Other data included relatively high frequencies of aneuploidy in hypercellular adenomas (29%; six of 21 cases) and diploid status of carcinomas (36%; 12 of 33 cases). In summary, although the overall findings show a trend toward increasing aneuploidy from well-differentiated and hypercellular adenomas to minimally and widely invasive follicular carcinomas, the aneuploidy data are inconsistent and indicative of its nonspecificity and limited diagnostic usefulness.

Adrenal pseudocyst: a clinical and pathologic study of eight cases.

Adrenal pseudocysts are cystic lesions arising within the adrenal gland surrounded by a fibrous tissue wall devoid of a recognizable lining layer. This study comprised eight adrenal pseudocysts surgically excised at the Massachusetts General Hospital. The median age of the patients was 41 years. Seven of the eight individuals were women. There was no apparent etiologic relationship to prior trauma or pregnancy. Half of the patients described symptoms that resolved following pseudocyst removal. The remaining individuals were asymptomatic with adrenal pseudocysts discovered incidentally during the work up of other medical problems. Adrenal pseudocyst size ranged from 1.8 to 10 cm. Pseudocyst size did not correlate with the presence of symptoms. The histologic and immunohistochemical findings in this study suggest that at least some adrenal pseudocysts are of vascular origin. In two lesions, small foci of residual cells lining the inner pseudocyst wall were found that expressed factor VIII antigen (also Ulex Europaeus lectin in one case) but not vimentin, keratin, or epithelial membrane antigen. In both cases, abundant elastic tissue was present in the pseudocyst wall, and in one lesion, adrenal vein smooth muscle was present as well. In both cases, dilated sinusoids were found at the periphery of the pseudocysts and, in one lesion, the sinusoids appeared to coalesce to form the pseudocyst cavity. In the majority of the remaining six cases there was also some histologic evidence to suggest a vascular origin. In five and three cases, respectively, abundant elastic tissue and adrenal vein smooth muscle were found within the pseudocyst wall. In two lesions, both elastic tissue and smooth muscle were present. In addition, adrenal sinusoids were prominently dilated at the periphery of four pseudocysts and, in one case, the sinusoids appeared to coalesce to form the pseudocyst cavity.

Epithelial-lined (true) cyst of the adrenal gland: a case report.

A case of an epithelial-lined (true) adrenal cyst is reported. Although over 300 adrenal cysts have been reported in the literature, true cysts are rare. In this case, a 4.0 cm cyst lined by cuboidal to flattened cells with bland cytologic features was incidentally found at autopsy. Immunologic studies performed on formalin-fixed, paraffin-embedded sections demonstrated that the cells expressed keratins (AE1/AE3+, CAM 5.2+, and MAK-6+) and were negative for epithelial membrane antigen, vimentin, factor VIII, and desmin. Normal adrenal cortical and medullary cells did not express keratins, suggesting that the cyst lining was not derived from either adrenal cortex or medulla. A mesothelial origin, with a pathogenesis analogous to the formation of primary cysts of the spleen, is proposed.

Diethylcarbamazine-mediated clearance of Brugia pahangi microfilariae in immunodeficient nude mice.

Congenitally athymic nude C3H/HeN mice, microfilaremic with Brugia pahangi, were treated with diethylcarbamazine citrate (DEC). A single oral dose (100 mg/Kg body weight) of DEC resulted in the rapid reduction of numbers of circulating microfilariae in nude, thymus-grafted nude and complement-depleted nude mice. Antibodies of the IgM and IgG isotypes were not detected in the serum of microfilaremic nudes or on the microfilarial surface. These results suggest that DEC-mediated clearance of microfilariae from the circulation of nude mice is probably independent of thymus-dependent immunological mechanisms.