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Aims: To estimate prevalence of diagnosed (dDM) and undiagnosed diabetes (uDM) in Hungary and investigate determinants of uDM., Methods: Data was obtained from the nationally representative H-UNCOVER study. As laboratory measurements were available for 11/19 Hungarian counties, n = 5,974/17,787 people were eligible. After exclusions, 5,673 (representing 4,976,097 people) were included. dDM was defined by self-reporting, while uDM as negative self-reporting and elevated fasting glucose (≥7 mmol/l) and/or HbA1c (≥48 mmol/mol). Logistic regression for complex samples was used to calculate comparisons between dDM and uDM adjusted for age and BMI., Results: Diabetes prevalence was 12.0 %/11.9 % (women/men, 95 %CI:10.7-13.4 %/10.7-13.2 %), while 2.2 %/2.8 % (1.7-2.8 %/2.2-3.6 %) of women/men were uDM. While the proportion of uDM vs. dDM was similar for women ≥ 40, men in their forties had the highest odds for uDM. Neither unemployment (women/men OR:0.58 [0.14-2.45]/0.50 [0.13-1.92]), nor education level (tertiary vs. primary; women/men OR: 1.16 [0.53-2.56]/ 0.53 [0.24-1.18]) were associated with uDM. The risk of uDM was lower in both sexes with chronic morbidities., Conclusions: We report higher prevalence of diabetes and undiagnosed diabetes than previous Hungarian estimates. The finding that socioeconomic factors are not associated to uDM suggests that universal health care could provide equitable access to diabetes diagnosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Cardiovascular autonomic neuropathy (CAN) is a frequent complication of diabetes mellitus and is associated with increased morbidity and mortality in patients with diabetes. Hence, early and correct diagnosis of CAN is crucial. Standard cardiovascular reflex rests (CARTs) have been the gold standard of CAN assessment. Originally, CARTs consisted of five reflex tests, but measuring diastolic blood pressure response to sustained handgrip exercise has no longer been suggested as an established clinical test. Increasing body of evidence suggests that isometric handgrip test should no longer be used for the evaluation of sympathetic dysfunction during cardiovascular autonomic neuropathy assessment in diabetic patients. The associations of isometric handgrip test results with parameters of hypertension and markers of hypertension-related target-organ damage in diabetic and non-diabetic individuals point toward its potential role as a screening tool to identify patients with high cardiovascular risk. The current review summarizes historical view of standard cardiovascular reflex tests and latest data on isometric handgrip test., Competing Interests: Declaration of competing interest The authors have no conflicting interest to declare., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Introduction: Autonomic and sensory neuropathy have been observed in both prediabetes and manifest diabetes mellitus. However, there is a lack of available data regarding whether patients at a moderate or high risk of developing diabetes, yet without a current diagnosis of prediabetes or diabetes, exhibit an increased prevalence of neuropathy., Methods: FINDRISC (Finnish Diabetes Risk Score) was used to classify individuals at risk (≥12 points, n = 44; control <12 points, n = 28). HbA1c levels >5.6% served as exclusion criteria, and patients with known medical conditions predisposing to neuropathy were also excluded. Cardiac autonomic function (Ewing tests) and peripheral sensory neuropathy (Neurometer and Q-sense) were assessed by standardized protocols, and their potential association with increased FINDRISC points was analyzed using a regression model., Results: Mean age was 46.7 ± 14.3 years in the control and 55.7 ± 14.1 years in the increased risk group. Male/female ratio did not differ. Individuals with increased risk of diabetes were more obese (BMI: 29.9 ± 12.5 kg/m 2 vs. 25.9 ± 8.9 kg/m 2 ). Additionally, hypertension was more frequent among them (68.2% vs. 17.9%), and their lipid parameters were also less favorable. Parasympathetic neuropathy was present in both groups (56.8% vs. 32.1%, respectively). Sympathetic neuropathy was not found. Sensory nerve dysfunction was of low prevalence in the high-risk group and did not occur in healthy controls. In multiple logistic regression analysis, HbA1c exhibited an independent association with parasympathetic neuropathy (OR: 5.9; 95% CI: 1.08-32.68; p < 0.041)., Discussion: An increased risk of developing prediabetes/diabetes does not appear to have a strong correlation with an increased likelihood of developing autonomic or sensory neuropathy. However, the etiology behind the occurrence of parasympathetic autonomic neuropathy in healthy individuals remains unknown., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Körei, Békeffy, Menyhárt, Osgyán, Istenes, Horváth and Kempler.)

Introduction: In 1989, the St Vincent declaration aimed to approximate pregnancy outcomes of diabetes to that of healthy pregnancies. We aimed to compare frequency and trends of outcomes of pregnancies affected by type 1 diabetes and controls in 1996-2018., Methods: We used anonymized records of a mandatory nation-wide registry of all deliveries between gestational weeks 24 and 42 in Hungary. We included all singleton births (4,091 type 1 diabetes, 1,879,183 controls) between 1996 and 2018. We compared frequency and trends of pregnancy outcomes between type 1 diabetes and control pregnancies using hierarchical Poisson regression., Results: The frequency of stillbirth, perinatal mortality, large for gestational age, caesarean section, admission to neonatal intensive care unit (NICU), and low Appearance, Pulse, Grimace, Activity, and Respiration (APGAR) score was 2-4 times higher in type 1 diabetes compared to controls, while the risk of congenital malformations was increased by 51% and SGA was decreased by 42% (all p<0.05). These observations remained significant after adjustment for confounders except for low APGAR scores. We found decreasing rate ratios comparing cases and controls over time for caesarean sections, low APGAR scores (p<0.05), and for NICU admissions (p=0.052) in adjusted models. The difference between cases and controls became non-significant after 2009. No linear trends were observed for the other outcomes., Conclusions: Although we found that the rates of SGA, NICU care, and low APGAR score improved in pregnancies complicated by type 1 diabetes, the target of the St Vincent Declaration was only achieved for the occurrence of low APGAR scores., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Fazekas-Pongor, Svébis, Major, Pártos, Dósa, Mészáros, Horváth, Domján, Zsirai and Tabák.)

Objectives: Our aim in this study was to compare the efficacy and safety of commercially available fixed-ratio combinations (FRCs) of glucagon-like peptide-1 receptor agonists (GLP-1RAs) and basal insulins by a network meta-analysis of randomized controlled trials (RCTs) of people with type 2 diabetes., Methods: We present a systematic review and network meta-analyses of RCTs of individuals with type 2 diabetes randomized to FRCs or to their components for ≥24 weeks. All reports were obtained from PubMed or ClinicalTrials.gov up to February 28, 2022. The primary outcome was glycated hemoglobin (A1C) level attained. Secondary outcomes included fasting plasma glucose, change in body weight, and incident hypoglycemia. Treatment effects were estimated as mean difference (MD) and standard error (SE), or as odds ratio (OR) with 95% confidence interval (CI) using the fixed combination of insulin glargine 100 IU/mL and lixisenatide (iGlarLixi) as reference., Results: We included 29 RCTs from among the 1,404 articles identified. No direct comparisons between FRCs were found. After excluding some insulin-capped trials to reach model consistency, both FRCs were more efficacious regarding A1C than their components, but no difference between FRCs was found (MD, -0.10%; SE, 0.10%). The effect of the fixed combination of insulin degludec and liraglutide (IDegLira) (MD, -0.47 mmol/L; SE, 0.24 mmol/L) and basal insulins was similar to that of iGlarLixi (reference) on fasting glucose, whereas GLP-1RAs had lower efficacy than iGlarLixi. Weight gain was lower with GLP-1RAs and IDegLira (MD, -0.72 kg; SE, 0.32 kg) than with iGlarLixi (reference) and higher with basal insulins. Incident hypoglycemia (based on different definitions) was least frequent with GLP-1RAs, followed by IDegLira (OR, 0.78; 95% CI, 0.39 to 1.57), iGlarLixi (reference), and basal insulins., Conclusions: For A1C, both FRCs were more efficacious over their individual components, with similar efficacies of the 2 FRCs., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

It is well accepted that COVID-19-related mortality shows a strong age dependency. However, temporal changes in the age distribution of excess relative mortality between waves of the pandemic are less frequently investigated. We aimed to assess excess absolute mortality and the age-distribution of all-cause mortality during the second and third waves of the COVID-19 pandemic in Hungary compared to the same periods of non-pandemic years. Rate ratios for excess all-cause mortality with 95% confidence intervals and the number of excess deaths for the second (week 41 of 2020 through week 4 of 2021) and third waves (weeks 7-21 of 2021) of the COVID pandemic for the whole of Hungary compared to the same periods of the pre-pandemic years were estimated for 10-year age strata using Poisson regression. Altogether, 9771 (95% CI: 9554-9988) excess deaths were recorded during the second wave of the pandemic, while it was lower, 8143 (95% CI: 7953-8333) during the third wave. During the second wave, relative mortality peaked for ages 65-74 and 75-84 (RR 1.37, 95%CI 1.33-1.41, RR 1.38, 95%CI 1.34-1.42). Conversely, during the third wave, relative mortality peaked for ages 35-44 (RR 1.43, 95%CI 1.33-1.55), while those ≥65 had substantially lower relative risks compared to the second wave. The reduced relative mortality among the elderly during the third wave is likely a consequence of the rapidly increasing vaccination coverage of the elderly coinciding with the third wave. The hugely increased relative mortality of those 35-44 could point to non-biological causes, such as less stringent adherence to non-pharmaceutical measures in this population., (© 2022. The Author(s), under exclusive licence to American Aging Association.)

Background: People with diabetic cardiovascular autonomic neuropathy (CAN) have increased cardiovascular mortality. However, the association between distal symmetric polyneuropathy (DSPN) or CAN with all-cause mortality is much less investigated. Thus, we set out to examine the effect of CAN and DSPN on all-cause mortality in a well-phenotyped cohort., Methods: All diabetes cases ( n = 1,347) from the catchment area of a secondary diabetes care centre who had medical examination including neuropathy assessment between 1997 and 2016 were followed up for all-cause mortality in the NHS Hungary reimbursement database until 2018. We investigated the association of CAN (Ewing tests) and DSPN (Neurometer) with all-cause mortality using Cox models stratified by diabetes type., Results: Altogether, n = 131/1,011 persons with type 1/type 2 diabetes were included. Of the participants, 53%/43% were male, mean age was 46 ± 12/64 ± 10 years, diabetes duration was 13 ± 10/7 ± 8 years, 42%/29% had CAN, and 39%/37% had DSPN. During the 9 ± 5/8 ± 5-year follow-up, n = 28/494 participants died. In fully adjusted models, participants with type 1 diabetes patients with versus without DSPN had an increased mortality (HR 2.99, 95% CI 1.4-8.63), while no association with CAN was observed. In type 2 diabetes, both DSPN and CAN independently increased mortality (HR 1.32, 95% CI: 1.07-1.64, and HR 1.44, 95% CI: 1.17-1.76)., Conclusions: Our results are compatible with an increased risk of mortality in people with type 1 diabetes and DSPN. Furthermore, we report a similarly strong association between DSPN and CAN and all-cause mortality in type 2 diabetes mellitus., Competing Interests: The authors declare that that they have no competing interests., (Copyright © 2021 Orsolya E. Vági et al.)

The distinction between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-related and community-acquired pneumonias poses significant difficulties, as both frequently involve the elderly. This study aimed to predict the risk of SARS-CoV-2-related pneumonia based on clinical characteristics at hospital presentation. Case-control study of all patients admitted for pneumonia at Semmelweis University Emergency Department. Cases (n = 30) were patients diagnosed with SARS-CoV-2-related pneumonia (based on polymerase chain reaction test) between 26 March 2020 and 30 April 2020; controls (n = 82) were historical pneumonia cases between 1 January 2019 and 30 April 2019. Logistic models were built with SARS-CoV-2 infection as outcome using clinical characteristics at presentation. Patients with SARS-CoV-2-related pneumonia were younger (mean difference, 95% CI: 9.3, 3.2-15.5 years) and had a higher lymphocyte count, lower C-reactive protein, presented more frequently with bilateral infiltrate, less frequently with abdominal pain, diarrhoea, and nausea in age- and sex-adjusted models. A logistic model using age, sex, abdominal pain, C-reactive protein, and the presence of bilateral infiltrate as predictors had an excellent discrimination (AUC 0.88, 95% CI: 0.81-0.96) and calibration (p = 0.27-Hosmer-Lemeshow test). The clinical use of our screening prediction model could improve the discrimination of SARS-CoV-2 related from other community-acquired pneumonias and thus help patient triage based on commonly used diagnostic approaches. However, external validation in independent datasets is required before its clinical use.

Objective: Monophasic glucose response (MGR) during an oral glucose tolerance test (OGTT) and gestational diabetes mellitus (GDM) are predictors of type 2 diabetes mellitus (T2DM). We investigated the association between current MGR and (1) glucose tolerance during a pregnancy 3 years before and (2) current glucose tolerance status. We also sought (3) other determinants of MGR. Research Design and Methods . We conducted a nested case-control study of GDM ( n = 47 early GDM, diagnosed between 16 and 20 weeks of gestation; n = 40 late GDM, diagnosed between 24 and 28 weeks of gestation) and matched healthy controls ( n = 37, normal glucose tolerance during pregnancy) all free from diabetes at follow-up 3.4 ± 0.6 years after delivery. Glucose tolerance was determined by 2-hour 75 g OGTT. Monophasic and biphasic groups were defined based on serum glucose measurements during OGTT., Results: The biphasic group was younger, had lower triglyceride levels and area under the OGTT glucose curve, and was less frequently diagnosed with early GDM (25 vs. 45%, all p < 0.05). Women with a biphasic response also tended to have lower systolic blood pressure ( p < 0.1). No differences were found in fasting and 2-hour glucose and insulin levels, or BMI. According to multiple logistic regression, MGR was associated with prior early GDM (OR 2.14, 95% CI 0.92-4.99) and elevated triglyceride levels (OR 2.28, 95% CI 1.03-5.03/log (mmol/l))., Conclusions: We found that more severe, early-onset GDM was an independent predictor of monophasic glucose response suggesting that monophasic response may represent an intermediate state between GDM and manifest type 2 diabetes., Competing Interests: The authors declare no conflict of interest (financial disclosure) with the findings of the article., (Copyright © 2020 Timea Tänczer et al.)

Objective: Historically, a set of 5 Cardiovascular Autonomic Reflex Tests (CARTs) were considered to be the gold standard in the assessment of Cardiovascular Autonomic Neuropathy (CAN). However, measuring diastolic Blood Pressure (BP) response to sustained handgrip is omitted in recent guidelines. We aimed to assess the association between the handgrip and the other 4 tests as well as to identify determinants of the handgrip test results in diabetic patients., Patients and Methods: 353 patients with diabetes (DM) were recruited (age: 60.2±7.4 years; female: 57.2%; BMI: 29.3±2.1 kg/m2; DM duration: 15.6±9.9 years; HbA1c: 7.8±1.4% (66 mmol/mol); with type 1 DM: 18.1%). CAN was assessed by 5 CARTs: the deep breathing test, Valsalva ratio, 30/15 ratio, handgrip and orthostatic hypotension test., Results: Sensitivity and specificity of the handgrip test in the diagnosis of definite CAN were 24.6% (95%CI 17.7-33.1%) and 79.4% (95%CI 73.3-84.4%), respectively. Results of the handgrip test did not show any association with those of the deep-breathing test (y=0.004, p=0.563), 30/15 ratio (y=0.282, p=0.357), Valsalva ratio (y=-0.058, p=0.436) and orthostatic hypotension (y=-0.026, p=0.833). Handgrip test abnormality showed an independent association with higher initial diastolic BP (OR 1.05, p=0.0009) and an independent inverse association with the presence of hypertension (OR=0.42, p=0.006)., Conclusion: Our data confirm that the handgrip test should no longer be part of the cardiovascular autonomic testing being highly dependent on hypertensive status and baseline diastolic BP. Exaggerated exercise pressor response is proposed as putative mechanism for the inverse association between abnormal results of the handgrip test and hypertension. Adequate CARTs are important to allow their use in clinical trials and for the prevention of DM-associated complications by initiating early treatment.

In contrast to gastric dysfunction, diabetes-related functional impairments of the small and large intestine have been studied less intensively. The gastrointestinal tract accomplishes several functions, such as mixing and propulsion of luminal content, absorption and secretion of ions, water, and nutrients, defense against pathogens, and elimination of waste products. Diverse functions of the gut are regulated by complex interactions among its functional elements, including gut microbiota. The network-forming tissues, the enteric nervous system) and the interstitial cells of Cajal, are definitely impaired in diabetic patients, and their loss of function is closely related to the symptoms in diabetes, but changes of other elements could also play a role in the development of diabetes mellitus-related motility disorders. The development of our understanding over the recent years of the diabetes-induced dysfunctions in the small and large intestine are reviewed in this article.

Among their beneficial effects, non-steroidal anti-inflammatory drugs may also exert several side effects which depend on the dosage and the type of these medications. The most frequent gastrointestinal side effects usually develop shortly after the beginning of their administration, but others such as cardiovascular interactions (which are present much less frequently than gastrointestinal side effects) can also occur after the beginning of drug administration without a latency period. For a long-term treatment, non-steroidal anti-inflammatory drugs are most frequently used in the elderly population where patients typically have high cardiovascular risk and take other medicines, e.g. low dose acetylsalicylic acid that can interact with non-steroidal anti-inflammatory drugs; in this aspect diclofenac may cause less side effects. In this review, the authors briefly review cardiovascular side effects of non-steroidal anti-inflammatory drugs, the processes which potentially influence them, therapeutic consequences and their interaction with acetylsalicylic acid.

The regulation of gastrointestinal motility mainly involves the smooth muscle, neural (extrinsic and intrinsic), and hormonal elements, the glial cells, and the interstitial cells of Cajal. An orchestrated function of all these components is required for the appropriate propulsive movement of the food in the gastrointestinal tract. Gastroparesis, a pathological slowing-down of gastric emptying, is a result of the damage to the tissue elements involved in the regulation of motility. Gastroparesis is one of the well-known complications of long-standing diabetes mellitus. Although it is rarely a life-threatening complication, it has a deteriorating effect on the quality of life, leads to unpredictable oscillation of the blood glucose level, and increases the time required for the absorption of food and medicines. This review describes the clinical characteristics of diabetic gastroparesis and summarizes the organic and functional motility abnormalities caused by this complication. Finally, the currently available and potential future therapeutic approaches are summarized.

In situ hybridization, quantitative reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry, and Western blot analysis were applied to study the changes in expression of the major nociceptive ion channel transient receptor potential vanilloid type 1 receptor (TRPV1) after the perineural application of capsaicin or nerve transection. In control rats, quantitative morphometric and statistical analyses of TRPV1 protein and mRNA expression in L5 dorsal root ganglion cells revealed distinct populations of small (type C) and small-to-medium (type B) neurons, which showed very high and moderate levels of TRPV1, whereas larger (type A) neurons mostly did not express this receptor. After either transection or capsaicin treatment of the sciatic nerve, immunohistochemistry and Western blotting demonstrated a massive (up to 80%) decrease in the proportion of TRPV1-immunoreactive neurons and TRPV1 protein at all postoperative survival times. In situ hybridization indicated marked decreases (up to 85%) in the proportion of neurons that expressed TRPV1 mRNA after sciatic nerve transection. In contrast, although perineural treatment with capsaicin resulted in similar substantial decreases in the proportions of type B and C neurons of the L5 dorsal root ganglia 3 days postoperatively, a clear-cut tendency to recovery was observed thereafter. Hence, the proportions of both type B and C neurons expressing TRPV1 mRNA reached up to 70% of the control levels at 30 days postoperatively. In accord with these findings, quantitative RT-PCR revealed a marked and significant recovery in TRPV1 mRNA after perineural capsaicin but not after nerve transection. These observations suggest the involvement of distinct cellular mechanisms in the regulation of the TRPV1 mRNA expression of damaged neurons, specifically triggered by the nature of the injury. The present findings imply that the antinociceptive and anti-inflammatory effects of perineurally applied capsaicin involve distinct changes in neuronal TRPV1 mRNA expression and long-lasting alterations in (post)translational regulation., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

Background & Aims: Gastrointestinal stromal tumors (GIST) are related to interstitial cells of Cajal (ICC) and often contain activating stem cell factor receptor (Kit) or platelet-derived growth factor receptor alpha (Pdgfra) mutations. Kit/Pdgfra inhibitors such as imatinib mesylate have increased progression-free survival in metastatic GIST but are not curative. In mouse models we investigated whether Kit(low) ICC progenitors could represent an inherently Kit/Pdgfra inhibitor-resistant reservoir for GIST., Methods: Isolated Kit(low)Cd44(+)Cd34(+) cells were characterized after serial cloning. The tumorigenic potential of spontaneously transformed cells was investigated in nude mice. The Kit(low)Cd44(+)Cd34(+) cells' responsiveness to Kit activation and blockade was studied by enumerating them in Kit(K641E) mice (a GIST model), in mice with defective Kit signaling, and pharmacologically., Results: Single isolated Kit(low)Cd44(+)Cd34(+) cells were clonogenic and capable of self-renewal and differentiation into ICC. In nude mice, spontaneously transformed cells formed malignant tumors expressing GIST markers. The Kit(low)Cd44(+)Cd34(+) cells were resistant to in vitro Kit blockade, including by imatinib, and occurred in normal numbers in mice with reduced Kit signaling. In Kit(K641E) mice, the mutant ICC stem cells were grossly hyperplastic but remained imatinib-resistant. In contrast, the cancer stem, cell-targeting drug salinomycin blocked the proliferation of Kit(low)Cd44(+)Cd34(+) cells and increased their sensitivity to imatinib., Conclusions: Kit(low)Cd44(+)Cd34(+) progenitors are true stem cells for normal and hyperplastic ICC and give rise to GIST. Resistance to Kit/Pdgfra inhibitors is inherent in GIST and is caused by the native ICC stem cells' lack of dependence on Kit for survival, which is maintained after the acquisition of oncogenic Kit mutation. Cancer stem cell drugs may target these cells., (Copyright © 2010 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background & Aims: Maintaining the integrity of networks of interstitial cells of Cajal (ICC) is essential to preserve orderly contractile activity and neuroregulation in the gastrointestinal tract and to restore these functions after tissue damage or surgeries. Maintenance of ICC requires insulin-dependent or insulin-like growth factor I (IGF-I)-dependent production of membrane-bound stem cell factor (SCF) and may involve regeneration from local progenitors. Our goal was to identify ICC precursors in postnatal murine gastric muscles., Methods: We used flow cytometry and immunohistochemistry to examine freshly dissected and cultured muscles for cells expressing CD34, an adhesion molecule expressed by stromal tumors; CD44, which occurs on mesenchymal stem cells; and receptors for SCF (Kit), insulin (Insr), and IGF-I (Igf1r). Slow waves were studied by intracellular recording., Results: In gastric muscles, we identified rare, Kit(low)CD44(+)CD34(+)Insr(+)Igf1r(+) cells resembling common embryonic precursors of ICC and smooth muscle. These putative progenitors were absent from organotypic cultures lacking mature ICC (Kit(+)CD44(+)CD34(-)Insr(-)Igf1r(-)) due to prolonged insulin/IGF-I deprivation but were rescued by IGF-I that also prevented ICC loss. Soluble SCF failed to prevent the loss of mature ICC but dramatically expanded the putative progenitors, which supported robust slow wave activity despite retaining an immature, Kit(+)CD44(+)CD34(+)Insr(+)Igf1r(+) phenotype. Differentiation of these cells into mature, network-forming ICC required IGF-I. Conversely, restoration of ICC networks by IGF-I after prolonged insulin and IGF-I deprivation required the survival of the presumed progenitors., Conclusions: Kit(low)CD44(+)CD34(+)Insr(+)Igf1r(+) cells may be local progenitors for gastric ICC and stromal tumors. Loss of these cells may contribute to gastrointestinal dysmotilities.

Background & Aims: Diabetic gastroparesis involves neuropathy, myopathy, and depletion of interstitial cells of Cajal (ICC), which may cause dysrhythmias and impaired neural control. Most murine gastric ICC depend on stem cell factor (SCF) signaling but can also be maintained with insulin or insulin-like growth factor-I (IGF-I). We investigated whether SCF could mediate the actions of insulin and IGF-I., Methods: Expression of insulin receptor, IGF-I receptor, and SCF was studied in gastric muscles and purified ICC by immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). The effects of insulin/IGF-I deficiency on SCF, ICC, smooth muscle, and neurons were investigated in nonobese diabetic mice and organotypic cultures by immunohistochemistry, microarrays, and/or quantitative RT-PCR. ICC in organotypic cultures were also studied after immunoneutralization of endogenous SCF., Results: Insulin and IGF-I receptors were detected in smooth-muscle cells and myenteric neurons but not in ICC. Cell-surface expression of SCF was only found in smooth-muscle cells. ICC depletion in diabetes was accompanied by smooth-muscle atrophy and reduced SCF, whereas neuron-specific gene expression remained unchanged. In organotypic cultures, prevention of ICC loss by insulin or IGF-I was paralleled by rescue of smooth-muscle cells and SCF expression but not of myenteric neurons. Immunoneutralization of endogenous SCF caused ICC depletion closely resembling that elicited by insulin/IGF-I deficiency., Conclusions: Reduced insulin/IGF-I signaling in diabetes may lead to ICC depletion and its consequences by causing smooth-muscle atrophy and reduced SCF production. Thus, myopathy may play a more central role in diabetic gastroenteropathies than previously recognized.

Damage to interstitial cells of Cajal (ICC), pacemakers, and mediators of neuromuscular neurotransmission in the gastrointestinal tract contributes to the pathogenesis of diabetic gastroenteropathy in both patients and animal models. ICC depletion in diabetes may result from chronic hyperglycemia or lost/ineffective insulin signaling. Because independent control of insulin and glucose concentrations is difficult in chronic in vivo studies, we used long-term organotypic cultures to address this problem. Murine gastric muscles were cultured in normoglycemic or hyperglycemic basal media with or without insulin or IGF-I for 1-3 months, the time required for gastroparesis and ICC damage to develop in diabetic mice. ICC were assessed by c-Kit immunohistochemistry and quantitative analysis of c-kit expression. Electrical pacemaking was studied by intracellular recording of slow waves. ICC survived for at least 34 days in unsupplemented normoglycemic media, but their networks, c-kit expression, and slow waves were profoundly reduced after 68 days. These changes could be entirely prevented by insulin or IGF-I supplementation. ICC networks were completely resistant to hyperglycemia for at least 72 days. Thus, hyperglycemia is unlikely to be responsible for the diabetes-associated depletion of ICC. In contrast, maintenance of ICC requires insulin or IGF-I, which are reduced or ineffective in diabetes.

Background: Interstitial cells of Cajal (ICCs) are mesenchymal cells that play critical roles in gastrointestinal motility as electrical pacemakers and mediators of neuromuscular neurotransmission. Although depletions of ICCs have been implicated in several gastrointestinal motor disorders, quantification of these cells has been difficult due to their varied morphology, regionally changing network density, and overall scarcity. Our goal was to evaluate flow cytometry (FCM) for the enumeration of ICCs., Methods: We identified murine ICCs in live gastrointestinal muscles or primary cell cultures grown in the presence or absence of stem cell factor (SCF)-expressing STO fibroblasts with fluorescent Kit (CD117) antibodies. Because this technique also labels resident macrophages nonspecifically, we identified the latter with additional fluorescent antibodies. Dispersed cells were analyzed by FCM., Results: ICCs represented 1.63 +/- 0.17% of the total cell count in the distal stomach (n = 18 mice) and 5.85 +/- 0.84% in the proximal colon and 6.28 +/- 0.61% in the distal colon (n = 3 mice). In fundic muscles of W/WV mice (n = 5) that virtually lack ICCs, very few Kit+ cells were detected. FCM identified approximately 2.6- to 7.3-fold more Kit+ ICCs in small intestinal cell cultures grown on STO fibroblasts expressing membrane-bound SCF (n = 6) than in cultures stimulated with soluble SCF (n = 6)., Conclusions: FCM is a sensitive and specific method for the unbiased quantification of ICCs., (2004 Wiley-Liss, Inc.)

Interstitial cells of Cajal (ICC) in the gastrointestinal tract generate and propagate slow waves and mediate neuromuscular neurotransmission. Although damages to ICC have been described in several gastrointestinal motor disorders, analysis of their gene expression in health and disease has been problematic because of the difficulties in isolating these cells. Our goal was to develop techniques for large-scale purification of ICC. Murine ICC were identified in live gastrointestinal muscles with fluorescent Kit antibodies. Because this technique also labels resident macrophages nonspecifically, we attempted to separate ICC from these cells by fluorescence-activated cell sorting with or without immunomagnetic presorting. Efficacy and specificity of ICC purification were tested by quantitative RT-PCR of cell-specific markers. Fluorescence-based separation of small intestinal ICC from unlabeled cells and macrophages tagged with F4/80 antibodies yielded 30,000-40,000 cells and approximately 60-fold enrichment of c-kit mRNA. However, the macrophage marker CD68 was also enriched approximately 6-fold. Magnetic presorting of ICC did not significantly improve selectivity. After labeling contaminating cells with additional paramagnetic (anti-CD11b, -CD11c) and fluorescent antibodies (anti-CD11b) and depleting them by magnetic presorting, we harvested approximately 2,000-4,000 cells from single gastric corpus-antrum muscles and detected an approximately 30-fold increase in c-kit mRNA, no enrichment of mast cells, and an approximately 4-fold reduction of CD68 expression. Adding labeled anti-CD45 antibody to our cocktail further increased c-kit enrichment and eliminated mast cells and macrophages. Smooth muscle cells and myenteric neurons were also depleted. We conclude that immunofluorescence-based sorting can yield ICC in sufficiently high numbers and purity to permit detailed molecular analyses.