A novel secretin receptor splice variant potentially useful for early diagnosis of pancreatic carcinoma

BACKGROUND ; AIMS: Pancreatic and bile duct carcinomas represent highly aggressive malignancies that evolve from secretin receptor-rich ductular cells. With premessenger RNA splicing abnormalities common in cancer, we evaluated whether an abnormal secretin receptor spliceoform were present, characterized it, and developed a serum assay for it. METHODS: Cancer cell lines and healthy and neoplastic tissue were studied by nested reverse-transcription polymerase chain reaction and sequencing. A promising spliceoform was isolated and characterized, and monoclonal antibodies were raised to 2 distinct regions. A dual antibody enzyme-linked immunosorbent assay was developed and applied to blinded serum samples from 26 patients with pancreatic carcinoma, 10 patients with chronic pancreatitis, and 14 controls. RESULTS: Each of 9 pancreatic cancer specimens and no normal tissue expressed a secretin receptor variant with exons 3 and 4 deleted. This encoded a 111-residue peptide with its first 43 residues identical to wild-type receptor, but, subsequent to a shift in coding frame and early truncation, the next 68 residues were unique in the transcriptome/proteome. This nonfunctional soluble protein did not bind or signal in response to secretin and was secreted from transfected MiaPaCa-2 cells. Elevated serum levels of this variant were present in 69% of pancreatic cancer patients, 60% of chronic pancreatitis patients, and 1 of 14 controls. CONCLUSIONS: We identified a novel abnormal spliceoform of the secretin receptor in pancreatic and bile duct cancers and developed a dual antibody sandwich enzyme-linked immunosorbent assay to measure it in the circulation. Initial application of this assay in patients with pancreatic cancer and chronic pancreatitis was promising, but additional validation will be required to evaluate its clinical utility.

Mumps

Mumps is a common childhood infection caused by the mumps virus. The hallmark of infection is swelling of the parotid gland. Aseptic meningitis and encephalitis are common complications of mumps together with orchitis and oophoritis, which can arise in adult men and women, respectively; other complications include deafness and pancreatitis. Clinical diagnosis can be based on the classic parotid swelling; however, this feature is not present in all cases of mumps and can also occur in various other disorders. Laboratory diagnosis is based on isolation of virus, detection of viral nucleic acid, or serological confirmation (generally presence of IgM mumps antibodies). Mumps is vaccine-preventable, and one dose of mumps vaccine is about 80% effective against the disease. Routine vaccination has proven highly effective in reducing the incidence of mumps, and is presently used by most developed countries; however, there have been outbreaks of disease in vaccinated populations. In 2005, a large epidemic peaked in the UK, and in 2006 the American midwest had several outbreaks. In both countries, the largest proportion of cases was in young adults. In the UK, susceptible cohorts too old to have been vaccinated and too young to have been exposed to natural infections were the primary cause of the mumps epidemic. In the USA, effectiveness and uptake in combination appear not to have been sufficient to obtain herd immunity for mumps in populations such as college students.

The role of common single-nucleotide polymorphisms on exon 9 and exon 12 skipping in nonmutated CFTR alleles

Classic cystic fibrosis (CF) is caused by two loss-of-function mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, whereas patients with nonclassic CF have at least one copy of a mutant gene that retains partial function of the CFTR protein. In addition, there are several other phenotypes associated with CFTR gene mutations, such as idiopathic chronic pancreatitis. In CFTR-associated disorders and in nonclassic CF, often only one CFTR mutation or no CFTR mutations can be detected. In this study, we screened 23 patients with CFTR-associated disorders for CFTR mutations by complete gene testing and quantitative transcript analysis. Mutations were found in 10 patients. In cells from respiratory epithelium, we detected aberrant splicing of CFTR mRNA in all investigated individuals. We observed a highly significant association between the presence of coding single-nucleotide polymorphisms (coding SNPs, or cSNPs) and increased skipping of exon 9 and 12. This association was found both in patients and in normal individuals carrying the same cSNPs. The cSNPs c.1540A>G, c.2694T>G, and c.4521G>A may have affected pre-mRNA splicing by changing regulatory sequence motifs of exonic splice enhancers, leading to lower amounts of normal transcripts. The analysis of CFTR exons indicated that less frequent and weak exonic splicing enhancer (ESE) motifs make exon 12 vulnerable to skipping. The number of splice variants in individuals with cSNPs was similar to previously reported values for the T5 allele, suggesting that cSNPs may enhance susceptibility to CFTR related diseases. In addition, cSNPs may be responsible for variation in the phenotypic expression of CFTR mutations. Quantitative approaches rather than conventional genomic analysis are required to interpret the role of cSNPs.

[Odynophagia of a young mother]

We report the case of a 34-year old mother presenting in the emergency unit because of fever and odynophagia. Herpetic esophagitis with Herpes simplex virus primoinfection in a immunocompetent patient was diagnosed. The virus was transmitted by her own child which came to be hospitalised because of herpetic stomatitis. In the blood chemistry pancreas enzymes were elevated which we interpreted as herpetic pancreatitis because of coincidence with herpetic esophagitis.

β-Glucan Antigenemia Anticipates Diagnosis of Blood Culture–Negative Intraabdominal Candidiasis

Rationale: Life-threatening intraabdominal candidiasis (IAC) occurs in 30 to 40% of high-risk surgical intensive care unit (ICU) patients. Although early IAC diagnosis is crucial, blood cultures are negative, and the role of Candida score/colonization indexes is not established. Objectives: The aim of this prospective Fungal Infection Network of Switzerland (FUNGINOS) cohort study was to assess accuracy of 1,3-β-d-glucan (BG) antigenemia for diagnosis of IAC. Methods: Four hundred thirty-four consecutive adults with abdominal surgery or acute pancreatitis and ICU stay 72 hours or longer were screened: 89 (20.5%) at high risk for IAC were studied (68 recurrent gastrointestinal tract perforation, 21 acute necrotizing pancreatitis). Diagnostic accuracy of serum BG (Fungitell), Candida score, and colonization indexes was compared. Measurements and Main Results: Fifty-eight of 89 (65%) patients were colonized by Candida; 29 of 89 (33%) presented IAC (27 of 29 with negative blood cultures). Nine hundred twenty-one sera were analyzed (9/patient): median BG was 253 pg/ml (46–9,557) in IAC versus 99 pg/ml (8–440) in colonization (P < 0.01). Sensitivity and specificity of two consecutive BG measurements greater than or equal to 80 pg/ml were 65 and 78%, respectively. In recurrent gastrointestinal tract perforation it was 75 and 77% versus 90 and 38% (Candida score ≥ 3), 79 and 34% (colonization index ≥ 0.5), and 54 and 63% (corrected colonization index ≥ 0.4), respectively. BG positivity anticipated IAC diagnosis (5 d) and antifungal therapy (6 d). Severe sepsis/septic shock and death occurred in 10 of 11 (91%) and 4 of 11 (36%) patients with BG 400 pg/ml or more versus 5 of 18 (28%, P = 0.002) and 1 of 18 (6%, P = 0.05) with BG measurement less than 400 pg/ml. β-Glucan decreased in IAC responding to therapy and increased in nonresponse. Conclusions: BG antigenemia is superior to Candida score and colonization indexes and anticipates diagnosis of blood culture–negative IAC. This proof-of-concept observation in strictly selected high-risk surgical ICU patients deserves investigation of BG-driven preemptive therapy.

ROLE OF PROSTAGLANDIN E2 IN THE REGULATION OF PANCREATIC STELLATE CELLS HYPER ACTIVITY ASSOCIATED WITH PANCREATIC CANCER

Pancreatic cancer is one of the most lethal type of cancer due to its high metastasis rate and resistance to chemotherapy. Pancreatic fibrosis is a constant pathological feature of chronic pancreatitis and the hyperactive stroma associated with pancreatic cancer. Strong evidence supports an important role of cyclooxygenase-2 (COX-2) and COX-2 generated prostaglandin E2 (PGE2) during pancreatic fibrosis. Pancreatic stellate cells (PSC) are the predominant source of extracellular matrix production (ECM), thus being the key players in both diseases. Given this background, the primary objective is to delineate the role of PGE2 on human pancreatic stellate cells (PSC) hyper activation associated with pancreatic cancer. This study showed that human PSC cells express COX-2 and synthesize high levels of PGE2. PGE2 stimulated PSC migration and invasion; expression of extra cellular matrix (ECM) genes and tissue degrading matrix metallo proteinases (MMP) genes. I further identified the PGE2 EP receptor responsible for mediating these effects on PSC. Using genetic and pharmacological approaches I identified the receptor required for PGE2 mediates PSC hyper activation. Treating PSC with Specific antagonists against EP1, EP2 and EP4, demonstrated that blocking EP4 receptor only, resulted in a complete reduction of PGE2 mediated PSC activation. Furthermore, siRNA mediated silencing of EP4, but not other EP receptors, blocked the effects of PGE2 on PSC fibrogenic activity. Further examination of the downstream pathway modulators revealed that PGE2 stimulation of PSC involved CREB and not AKT pathway. The regulation of PSC by PGE2 was further investigated at the molecular level, with a focus on COL1A1. Collagen I deposition by PSC is one of the most important events in pancreatic cancer. I found that PGE2 regulates PSC through activation of COL1A1 expression and transcriptional activity. Downstream of PGE2, silencing of EP4 receptor caused a complete reduction of COL1A1 expression and activity supporting the role of EP4 mediated stimulation of PSC. Taken together, this data indicate that PGE2 regulates PSC via EP4 and suggest that EP4 can be a better therapeutic target for pancreatic cancer to reduce the extensive stromal reaction, possibly in combination with chemotherapeutic drugs can further kill pancreatic cancer cells.

Chondroitin sulfate proteoglycan CSPG4 as a novel hypoxia-sensitive marker in pancreatic tumors.

CSPG4 marks pericytes, undifferentiated precursors and tumor cells. We assessed whether the shed ectodomain of CSPG4 (sCSPG4) might circulate and reflect potential changes in CSPG4 tissue expression (pCSPG4) due to desmoplastic and malignant aberrations occurring in pancreatic tumors. Serum sCSPG4 was measured using ELISA in test (n = 83) and validation (n = 221) cohorts comprising donors (n = 11+26) and patients with chronic pancreatitis (n = 11+20) or neoplasms: benign (serous cystadenoma SCA, n = 13+20), premalignant (intraductal dysplastic IPMNs, n = 9+55), and malignant (IPMN-associated invasive carcinomas, n = 4+14; ductal adenocarcinomas, n = 35+86). Pancreatic pCSPG4 expression was evaluated using qRT-PCR (n = 139), western blot analysis and immunohistochemistry. sCSPG4 was found in circulation, but its level was significantly lower in pancreatic patients than in donors. Selective maintenance was observed in advanced IPMNs and PDACs and showed a nodal association while lacking prognostic relevance. Pancreatic pCSPG4 expression was preserved or elevated, whereby neoplastic cells lacked pCSPG4 or tended to overexpress without shedding. Extreme pancreatic overexpression, membranous exposure and tissue(high)/sera(low)-discordance highlighted stroma-poor benign cystic neoplasm. SCA is known to display hypoxic markers and coincide with von-Hippel-Lindau and Peutz-Jeghers syndromes, in which pVHL and LBK1 mutations affect hypoxic signaling pathways. In vitro testing confined pCSPG4 overexpression to normal mesenchymal but not epithelial cells, and a third of tested carcinoma cell lines; however, only the latter showed pCSPG4-responsiveness to chronic hypoxia. siRNA-based knockdowns failed to reduce the malignant potential of either normoxic or hypoxic cells. Thus, overexpression of the newly established conditional hypoxic indicator, CSPG4, is apparently non-pathogenic in pancreatic malignancies but might mark distinct epithelial lineage and contribute to cell polarity disorders. Surficial retention on tumor cells renders CSPG4 an attractive therapeutic target. Systemic 'drop and restoration' alterations accompanying IPMN and PDAC progression indicate that the interference of pancreatic diseases with local and remote shedding/release of sCSPG4 into circulation deserves broad diagnostic exploration.

Stability of 3-bromotyrosine in serum and serum 3-bromotyrosine concentrations in dogs with gastrointestinal diseases

Abstract BACKGROUND: 3-Bromotyrosine (3-BrY) is a stable product of eosinophil peroxidase and may serve as a marker of eosinophil activation. A gas chromatography/mass spectrometry method to measure 3-BrY concentrations in serum from dogs has recently been established and analytically validated. The aims of this study were to determine the stability of 3-BrY in serum, to determine the association between peripheral eosinophil counts and the presence of an eosinophilic infiltrate in the gastrointestinal tract, and to compare serum 3-BrY concentrations in healthy dogs (n = 52) and dogs with eosinophilic gastroenteritis (EGE; n = 27), lymphocytic-plasmacytic enteritis (LPE; n = 25), exocrine pancreatic insufficiency (EPI; n = 26), or pancreatitis (n = 27). RESULTS: Serum 3-BrY concentrations were stable for up to 8, 30, and 180 days at 4°C, -20°C, and -80°C, respectively. There was no significant association between peripheral eosinophil count and the presence of eosinophils in the GI tissues (P = 0.1733). Serum 3-BrY concentrations were significantly higher in dogs with EGE (median [range] = 5.04 [≤0.63-26.26] μmol/L), LPE (median [range] = 3.60 [≤0.63-15.67] μmol/L), and pancreatitis (median [range] = 1.49 [≤0.63-4.46] μmol/L) than in healthy control dogs (median [range] = ≤0.63 [≤0.63-1.79] μmol/L; P < 0.0001), whereas concentrations in dogs with EPI (median [range] = 0.73 [≤0.63-4.59] μmol/L) were not different compared to healthy control dogs. CONCLUSIONS: The present study revealed that 3-BrY concentrations were stable in serum when refrigerated and frozen. No relationship between peripheral eosinophil count and the presence of eosinophils infiltration in the GI tissues was found in this study. In addition, serum 3-BrY concentrations were increased in dogs with EGE, but also in dogs with LPE and pancreatitis. Further studies are needed to determine whether measurement of 3-BrY concentrations in serum may be useful to assess patients with suspected or confirmed EGE or LPE.

Gallbladder and Pancreas in Henoch-Schönlein Purpura: Review of the Literature

OBJECTIVE Involvement of the pancreato-biliary system has been occasionally noted in Henoch-Schönlein purpura. Furthermore, cases of this vasculitis syndrome sometimes develop in the context of a viral hepatitis or after hepatitis vaccination. METHODS We completed a review of the literature. RESULTS Fifty reports published between 1977 and 2015 were retained for the analysis. A pancreato-biliary involvement was recognized in 34 individually well-described patients (♂:♀ = 19:15) with severe abdominal pain: pancreatitis (N = 20), acalculous cholecystitis (N = 11), both pancreatitis and cholecystitis (N = 3). In all of the pancreatitis patients, full recovery occurred (within ≤3 weeks in three-fourths of the patients). Cholecystectomy was performed in 8 cholecystitis patients. Seventeen Henoch-Schönlein patients (♂:♀ = 9:8) were associated with a viral liver disease and 4 (♂:♀ = 1:3) with a hepatitis vaccination. The vasculitis syndrome rapidly remitted in the 7 patients accompanying hepatitis A or E, in 2 patients of hepatitis B, and in the 4 patients preceded by a vaccination. Henoch-Schönlein purpura seemed to be serious in 5 patients with chronic hepatitis B and in 3 with chronic hepatitis C. CONCLUSIONS This analysis indicates that pancreato-biliary involvement is unusual in Henoch-Schönlein purpura. This complication deserves consideration in patients with especially severe abdominal pain. Finally, viral hepatitides and hepatitis vaccinations seem to be rare triggers of Henoch-Schönlein purpura.

Establishment of a complex inflammatory and protumorigenic microenvironment in mouse pancreas through cyclooxygenase-2 overexpression

Chronic inflammation is an established risk factor in the pathogenesis of many cancers. Pancreatic ductal adenocarcinoma, a malignancy with a particularly dismal prognosis, is no exception. Cyclooxygenase-2, a key enzyme induced by tissue injury, has a critical role in the generation of bioactive lipids known as prostaglandins. COX-2 overexpression is a frequent finding in pancreatic cancer, chronic pancreatitis and pancreatic intraepithelial neoplasias. To explore mechanisms through which chronic inflammation establishes and maintains a protumorigenic environment, we designed a mouse model overexpressing COX-2 in pancreatic parenchyma (BK5.COX-2 mice). We discovered that constitutive expression of COX-2 has a number of important sequelae, including upregulation of additional eicosanoid-generating enzymes and proinflammatory cytokines. Many of these molecular alterations precede the onset of significant histopathological changes. Increased levels of prostaglandins E2, D2, and F2α, 5-, 12-, and 15-hydroxyeiosatetraenoic acid (HETEs) were documented in tumors and pancreata of younger transgenic mice. Using a TaqMan™ Mouse Immune Panel, we detected elevated mRNAs for a number of proinflammatory cytokines (e.g., TNFα, IL-1β, IL-6). ^ Histological examination revealed early changes in the pancreas with similarities to human chronic pancreatitis, including loss of acinar cells, appearance of metaplastic ducts, and increased deposition of stroma. As the lesions progress, features typical of dysplastic and neoplastic cells emerged within the metaplastic ductal complexes, including cellular and nuclear atypia, crowding of cells, and loss of normal tissue architecture. The amount of fibroinflammatory stroma increased considerably; numerous small vessels were evident. A number of immunocytes from both the myeloid and lymphoid lineages were identified in transgenic pancreata. Neutrophils were the earliest to infiltrate, followed shortly by macrophages and mast cells. B and T cells generally began to appear by 8–12 weeks, and organized aggregates of lymphoid cells were often found in advanced lesions. ^ We tested the efficacy of several chemopreventive agents in this model, including celecoxib, a COX-2 selective inhibitor, pentoxifylline, a cytokine inhibitor, curcumin, a polyphenol with antioxidant and anti-inflammatory properties, and GW2974, a dual EGFR/ErbB2 inhibitor. Effects on lesion development were modest in the GW2974 and pentoxifylline treated groups, but significant prevention effects were observed with curcumin and celecoxib. ^

Calcium-dependent enzyme activation and vacuole formation in the apical granular region of pancreatic acinar cells

The pancreatic acinar cell produces powerful digestive enzymes packaged in zymogen granules in the apical pole. Ca2+ signals elicited by acetylcholine or cholecystokinin (CCK) initiate enzyme secretion by exocytosis through the apical membrane. Intracellular enzyme activation is normally kept to a minimum, but in the often-fatal human disease acute pancreatitis, autodigestion occurs. How the enzymes become inappropriately activated is unknown. We monitored the cytosolic Ca2+ concentration ([Ca2+]i), intracellular trypsin activation, and its localization in isolated living cells with specific fluorescent probes and studied intracellular vacuole formation by electron microscopy as well as quantitative image analysis (light microscopy). A physiological CCK level (10 pM) eliciting regular Ca2+ spiking did not evoke intracellular trypsin activation or vacuole formation. However, stimulation with 10 nM CCK, evoking a sustained rise in [Ca2+]i, induced pronounced trypsin activation and extensive vacuole formation, both localized in the apical pole. Both processes were abolished by preventing abnormal [Ca2+]i elevation, either by preincubation with the specific Ca2+ chelator 1,2-bis(O-aminophenoxy)ethane-N,N-N′,N′-tetraacetic acid (BAPTA) or by removal of external Ca2+. CCK hyperstimulation evokes intracellular trypsin activation and vacuole formation in the apical granular pole. Both of these processes are mediated by an abnormal sustained rise in [Ca2+]i.

Thymus transplantation, a critical factor for correction of autoimmune disease in aging MRL/+mice.

MRL/MP-+/+ (MRL/+) mice develop pancreatitis and sialoadenitis after they reach 7 months of age. Conventional bone marrow transplantation has been found to be ineffective in the treatment of these forms of apparent autoimmune disease. Old MRL/+ mice show a dramatic thymic involution with age. Hematolymphoid reconstitution is incomplete when fetal liver cells (as a source of hemopoietic stem cells) plus fetal bone (FB; which is used to recruit stromal cells) are transplanted from immunologically normal C57BL/6 donor mice to MRL/+ female recipients. Embryonic thymus from allogeneic C57BL/6 donors was therefore engrafted along with either bone marrow or fetal hematopoietic cells (FHCs) plus fragments of adult or fetal bone. More than seventy percent of old MRL/+ mice (> 7 months) that had been given a fetal thymus (FT) transplant plus either bone marrow or FHCs and also bone fragments survived more than 100 days after treatment. The mice that received FHCs, FB, plus FT from allogeneic donors developed normal T cell and B cell functions. Serum amylase levels decreased in these mice whereas they increased in the mice that received FHCs and FB but not FT. The pancreatitis and sialoadenitis already present at the time of transplantations were fully corrected according to histological analysis by transplants of allogeneic FHCs, FB and FT in the MRL/+ mice. These findings are taken as an experimental indication that perhaps stem cell transplants along with FT grafts might represent a useful strategy for treatment of autoimmune diseases in aged humans.

Colangiopancreatografia retrógrada endoscópica versus ultrassom endoscópico no diagnóstico histológico da estenose biliar maligna: revisão sistemática e metanálise

Introdução: O diagnóstico histológico das estenoses biliares é fundamental na definição da terapêutica a ser empregada, devido à heterogeneidade dos resultados dos estudos comparando o uso do escovado citológico e da biópsia transpapilar na colangiopancreatografia retrógada endoscópica (CPRE) com a punção aspirativa ecoguiada com agulha fina (ECO-PAAF) no diagnóstico histológico da estenose biliar maligna, e o fato de não existirem revisões sistemáticas e metanálises comparando esses métodos, este estudo propõe comparar esses dois métodos no diagnóstico histológico da estenose biliar maligna, através de revisão sistemática e metanálise da literatura. Métodos: Utilizando as bases de dados eletrônicas Medline, Embase, Cochrane, LILACS, CINAHL, e Scopus foram pesquisados estudos datados anteriormente a novembro de 2014. De um total de 1009 estudos publicados, foram selecionados três estudos prospectivos comparando ECO-PAAF e CPRE no diagnóstico histológico da estenose biliar maligna e cinco estudos transversais comparando ECO-PAAF com o mesmo padrão-ouro dos outros três estudos comparativos. Todos os pacientes foram submetidos ao mesmo padrão-ouro. Foram calculadas as variáveis do estudo (prevalência, sensibilidade, especificidade, valores preditivos positivos e negativos e acurácia) e realizada a metanálise utilizando os softwares Rev Man 5 e Meta-DiSc 1.4. Resultados: Um total de 294 pacientes foi incluído na análise. A probabilidade pré-teste para estenose biliar maligna foi de 76,66%. As sensibilidades médias da CPRE e da ECO-PAAF para o diagnóstico histológico da estenose biliar maligna foram de 49% e 76,5%, respectivamente; especificidades foram de 96,33% e 100%, respectivamente. As probabilidades pós-teste também foram determinadas: valores preditivos positivos de 98,33% e 100%, respectivamente, e valores preditivos negativos de 34% e 58,87%. As acurácias foram 60,66% e 82,25%, respectivamente. Conclusão: A ECO-PAAF é superior a CPRE com escovado citológico e/ou biópsia transpapilar no diagnóstico histológico da estenose biliar maligna. No entanto, um teste de ECO-PAAF ou CPRE com amostra histológica negativa não pode excluir a estenose biliar maligna, pois ambos os testes apresentam baixo valor preditivo negativo

Canine and Feline Diabetes Mellitus: Nature or Nurture?

There is evidence for the role of genetic and environmental factors in feline and canine diabetes. Type 2 diabetes is the most common form of diabetes in cats. Evidence for genetic factors in feline diabetes includes the overrepresentation of Burmese cats with diabetes. Environmental risk factors in domestic or Burmese cats include advancing age, obesity, male gender, neutering, drug treatment, physical inactivity, and indoor confinement. High-carbohydrate diets increase blood glucose and insulin levels and may predispose cats to obesity and diabetes. Low-carbohydrate, high-protein diets may help prevent diabetes in cats at risk such as obese cats or lean cats with underlying low insulin sensitivity. Evidence exists for a genetic basis and altered immune response in the pathogenesis of canine diabetes. Seasonal effects on the incidence of diagnosis indicate that there are environmental influences on disease progression. At least 50% of diabetic dogs have type 1 diabetes based on present evidence of immune destruction of P-cells. Epidemiological factors closely match those of the latent autoimmune diabetes of adults form of human type 1 diabetes. Extensive pancreatic damage, likely from chronic pancreatitis, causes similar to28% of canine diabetes cases. Environmental factors such as feeding of high-fat diets are potentially associated with pancreatitis and likely play a role in the development of pancreatitis in diabetic dogs. There are no published data showing that overt type 2 diabetes occurs in dogs or that obesity is a risk factor for canine diabetes. Diabetes diagnosed in a bitch during either pregnancy or diestrus is comparable to human gestational diabetes.