Biomedical subjects

P Puri

Publications and source records attributed to P Puri.

At least 127 records · Page 7Linked to original sources

PubMed1999

Abnormalities of elastin and elastic fibers in infantile hypertrophic pyloric stenosis.

Infantile hypertrophic pyloric stenosis (IHPS) is characterized by hypertrophy of the pyloric muscle, causing pyloric-channel narrowing and elongation. The rigidity of the muscle is increased, which is characterized as an "olive." Elastin is an insoluble protein that forms the major structural component of the elastic fibers and maintains the tensile strength of the tissues. To understand the possible histologic and molecular basis of the elasticity of the hypertrophic muscle in IHPS, we determined the distribution of the elastic fibers and elastin expression using Victoria blue van Gieson (VVG) staining and immunohistochemistry. In IHPS, the number of elastic fibers in the connective tissue was significantly increased in the thickened connective-tissue septa (CTS) compared with normal control specimens. In normal pyloric muscle, weak to moderate elastin immunoreactivity was observed in the CTS while no immunoreactivity was observed among the muscle fibers. In IHPS, strong immunoreactivity of elastin was observed in the CTS and moderate immunoreactivity among the hypertrophic smooth-muscle fibers. Our findings suggest that the increase on elastic fibers and elastin expression in the pyloric muscle in IHPS may play an important role in the development of pyloric-muscle rigidity, causing pyloric-canal obstruction.

Elasticity↗

PubMed1999

Biliary changes in extrahepatic portal venous obstruction: compression by collaterals or ischemic?

BACKGROUND: The postulated mechanisms of biliary abnormalities in extrahepatic portal venous obstruction (EHPVO) are either extrinsic compression by collaterals or ischemic injury due to venous thrombosis. If the former hypothesis is correct, then biliary changes should revert to normal after portasystemic shunt surgery. METHODS: Five patients with EHPVO who underwent portasystemic shunt surgery were studied. One of these patients had obstructive jaundice due to portal cavernoma. Endoscopic retrograde cholangiography (ERC) was performed before as well as after the shunt surgery. Doppler ultrasound and splenoportovenography were obtained to confirm the diagnosis of EHPVO as well as shunt patency. RESULTS: All patients had biliary abnormalities on pre-shunt ERC. The post-shunt ERC showed partial reversal of biliary abnormalities in 3 patients, complete reversal in 1 patient, and no reversal in 1 patient. Smooth strictures opened after shunt surgery and proximal dilatation disappeared in most patients. The indentations and caliber irregularities disappeared after shunt surgery, whereas angulations and ectasias of biliary ducts persisted. CONCLUSION: Shunt surgery results in regression of some of the biliary abnormalities and relieves biliary obstruction, suggesting that mechanical compression by collaterals is the mechanism behind biliary abnormalities in EHPVO. However, some biliary changes persist after shunt surgery signifying fixed obstruction due to ischemia or fibrous scarring. Thus, the two theories are not mutually exclusive.

Adolescent↗

PubMed1999

Altered endothelin-3 and endothelin-B receptor mRNA expression in Hirschsprung's disease.

BACKGROUND: Recently, the endothelin-3 (EDN3) and endothelin-B receptor (EDNRB) gene have been recognized as susceptibility genes for Hirschsprung's disease (HD). However, gene mutations have been observed only in limited cases, and the role of EDN3 in the pathogenesis and motility dysfunction in HD is not understood fully. To evaluate the possible implication of EDN3 and EDNRB for the development of HD, we examined the EDN3 and EDNRB mRNA level in bowel specimens of HD patients. METHODS: Entire resected specimens of colon were obtained from 14 patients with HD. Eight age-matched control patients without gastroenteric disorders also were examined. mRNA was extracted from ganglionic and aganglionic segments of the HD specimens and normal colons. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to evaluate the relative amount of EDN3 and EDNRB mRNA. RESULTS: In normal colon, constant EDN3 and EDNRB mRNA expression was observed. In HD, EDN3 and EDNRB mRNA expression was observed. In HD, EDN3 and EDNRB mRNA levels were decreased both in ganglionic and aganglionic segment in 2 cases. In 6 cases, EDN3 mRNA expression was decreased in aganglionic segment and in another 2 cases, EDNRB mRNA expression was decreased in aganglionic segment. In the remaining 4 cases, EDN3 and EDNRB mRNA levels were similar to controls. CONCLUSION: The authors' findings indicate that loss of EDN3 and EDNRB function may be involved in the maldevelopment of neural crest-derived cells causing HD in many patients.

Adolescent↗

PubMed1999

Increased tropoelastin and procollagen expression in the lung of nitrofen-induced diaphragmatic hernia in rats.

BACKGROUND/PURPOSE: Collagen and elastin, the predominant components of the lung connective tissue network, have been suggested to have an important influence on lung compliance and maximal expansion. Decrease in lung compliance and distensibility often is seen in human congenital diaphragmatic hernia (CDH) lung as well as in experimentally produced CDH lung. The aim of this study was to investigate mRNA levels of tropoelastin and alpha1 (I) procollagen, the precursors of elastin, and type I collagen, respectively, in CDH lung and to determine whether antenatal dexamethasone treatment has any effect on the production of these extracellular matrix proteins. METHODS: CDH model was induced in pregnant rats after administration of 100 mg nitrofen on day 9.5 of gestation (term, 22 days). Dexamethasone (0.25 mg/kg) was given on day 18.5 and 19.5. Cesarean section was performed on day 21. The fetuses were divided into three groups: group I, normal controls; group II, nitrofen-induced CDH; and group III, nitrofen-induced CDH with antenatal dexamethasone treatment. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to evaluate relative amounts of tropoelastin and alpha1 (I) procollagen mRNA. RESULTS: Levels of both tropoelastin and alpha1 (I) procollagen mRNA were significantly increased in group II compared with group I (P< .05). Neither tropoelastin nor alpha1 (I) procollagen mRNA levels were significantly different between group II and III. CONCLUSIONS: The increased local synthesis of tropoelastin and type I procollagen in CDH lung may be responsible for the increased rigidity and decreased compliance observed in the CDH hypoplastic lung. Glucocorticoids have no effect on pulmonary tropoelastin and alpha1 (I) procollagen gene expression in CDH lungs.

Animals↗

PubMed1999

Associated urological anomalies in children with unilateral renal agenesis.

PURPOSE: Unilateral renal agenesis has been noted in 1:1,000 autopsies. Recently an increased incidence of vesicoureteral reflux was reported in patients with a solitary kidney. We determined the incidence of associated renal abnormalities in children with unilateral renal agenesis. MATERIALS AND METHODS: We retrospectively reviewed 46 consecutive cases of unilateral renal agenesis diagnosed at our hospital between January 1985 and February 1998. Patient age at diagnosis ranged from newborn to 12.5 years (mean 2.8 years). There were 24 boys and 22 girls. The left kidney was absent in 27 patients and the right kidney was absent in the remaining 19. A total of 24 patients were evaluated for urinary tract infection and in the other 22 a solitary kidney was found during examination for congenital malformations, enuresis or abdominal pain. The diagnosis was made in all patients by abdominal ultrasound and confirmed by excretory urography, or diethylenetriaminepentaacetic acid or dimercapto-succinic acid scan. A voiding cystourethrogram was performed in 40 patients (87%). RESULTS: Associated urological anomalies were present in 22 of the 46 patients (48%) with unilateral renal agenesis, including primary vesicoureteral reflux in 13 (28%), ureterovesical junction obstruction in 5 (11%), ureteropelvic junction obstruction in 3 (7%), and ureterovesical and ureteropelvic junction obstruction in 1 (2%). Of the 22 patients 14 (64%) underwent surgical intervention. CONCLUSIONS: Nearly half of the patients with unilateral renal agenesis had associated urological anomalies. Vesicoureteral reflux was the most common associated anomaly and it was usually of high grade. Early recognition and treatment of urological anomalies in a patient with a solitary kidney are imperative to decrease the long-term risk of renal damage.

Child↗

PubMed1999

Granulocytic sarcoma of orbit preceding acute myeloid leukaemia: a case report.

A rare case of granulocytic sarcoma of orbit preceding the onset of acute meyloblastic leukaemia is presented. A 7-year-old boy presented with proptosis and no other systemic complaints. The appearance of an enlarged muscle in initial tomographic imaging with a normal peripheral blood picture caused a diagnostic problem. Biopsy suggested a diagnosis of granulocytic sarcoma. Subsequent bone marrow study revealed the diagnosis of acute myeloid leukaemia.

Child↗

PubMed1999

Notable postnatal alterations in the myenteric plexus of normal human bowel.

BACKGROUND: Nitric oxide is the most important transmitter in non-adrenergic non-cholinergic nerves in the human gastrointestinal tract. Impaired nitrergic innervation has been described in Hirschsprung's disease, hypertrophic pyloric stenosis, and intestinal neuronal dysplasia (IND). Recent findings indicate that hyperganglionosis, one of the major criteria of IND, is age dependent. However, information is scanty regarding the neurone density in normal human bowel in the paediatric age group. AIMS: To determine neurone density, morphology, and nitric oxide synthase distribution of the normal myenteric plexus at different ages during infancy and childhood. METHODS: Specimens were obtained from small bowel and colon in 20 children, aged one day to 15 years, at postmortem examination. Whole mount preparations were made of the myenteric plexus, which were subsequently stained using NADPH diaphorase histochemistry (identical to nitric oxide synthase) and cuprolinic blue (a general neuronal marker). The morphology of the myenteric plexus was described and the neurone density estimated. RESULTS: The myenteric plexus meshwork becomes less dense during the first years of life. The density of ganglion cells in the myenteric plexus decreases significantly with age during the first three to four years of life. The NADPH diaphorase positive (nitrergic) subpopulation represents about 34% of all neurones in the myenteric plexus. CONCLUSIONS: The notable decrease in neurone density in the myenteric plexus during the first years of life indicates that development is still an ongoing process in the postnatal enteric nervous system. Applied to the clinical situation, this implies that interpretation of enteric nervous system pathology is dependent on the age of the patient.

Adolescent↗

PubMed1999

Smooth muscle cell hypertrophy versus hyperplasia in infantile hypertrophic pyloric stenosis.

Infantile hypertrophic pyloric stenosis (IHPS) is characterized by hypertrophy of the pyloric muscle. It is not clearly understood whether pyloric muscle enlargement is due to hypertrophy (increase in cell size) or hyperplasia (increase in cell number). In the present study, we investigated proliferative activity as well as size and number of smooth muscle cells to understand the mechanism of pyloric muscle enlargement in IHPS. Full thickness muscle biopsy specimens were obtained from 18 IHPS patients at pyloromyotomy and from 11 age-matched controls. Formalin-fixed paraffin sections were immunostained with MAb MIB-1, which stains cells in the proliferating phase of the cell cycle. The proliferative index (PI) was calculated as the percentage of positive cell nuclei. Smooth muscle cell number per bundle and cell size were measured with an image analyzer. The mean PI in IHPS (9.6 +/- 5.7%) was significantly higher than that of controls (1.3 +/- 1.2%) (p < 0.01). There was a significant inverse correlation between PI and age at operation. Smooth muscle cell number per bundle in IHPS (240.6 +/- 129.4) was significantly greater than that of the controls (134.1 +/- 49.8) (p < 0.05). Smooth muscle cell size in IHPS (298.5 +/- 59.0 microm2) was also significantly greater than that of controls (154.3 +/- 21.5 microm2) (p < 0.01). Our findings suggest that hypertrophy-and hyperplasia as well-play important roles in increasing pyloric smooth muscle mass in IHPS.

Case-Control Studies↗

PubMed1999

Antenatal dexamethasone suppresses tumor necrosis factor-alpha expression in hypoplastic lung in nitrofen-induced diaphragmatic hernia in rats.

The hypoplastic lung in congenital diaphragmatic hernia (CDH) has both a quantitative and qualitative reduction in surfactant. Tumor necrosis factor-alpha (TNF-alpha) drastically decreases surfactant phospholipids synthesis by isolated human type II pneumocytes. Recently, it was shown that TNF-alpha mRNA expression is increased in human hypoplastic CDH lung. Antenatal glucocorticoid therapy demonstrates improved surfactant biochemical immaturity in an animal CDH model. The aim of this study was to investigate the effect of antenatal dexamethasone (Dex) on TNF-alpha protein and gene expression in nitrofen-induced CDH hypoplastic lung in rats. A CDH model was induced in pregnant rats after the administration of nitrofen on d 9.5 of gestation. Dex was given intraperitoneally on d 18.5 and 19.5. Cesarean section was performed on d 21. In situ hybridization was performed with a rat TNF-alpha-specific and digoxigenin-labeled oligonucleotide probe. TNF-alpha level was measured in solubilized lung tissue extracts by ELISA. In control lung, TNF-alpha mRNA expression was weak or absent. In contrast, strong TNF-alpha mRNA expression was demonstrated in type II pneumocytes and bronchiolar epithelium in CDH lung. In Dex-treated CDH lung, TNF-alpha mRNA expression was weak in both type II pneumocytes and the bronchiolar epithelium. The level of TNF-alpha was elevated significantly in CDH lung compared with levels in control lung extracts (p < 0.01). In Dex-treated CDH lung, TNF-alpha protein was significantly decreased compared with CDH lung (p < 0.05). Our findings suggest that the reduction in the local production of TNF-alpha may be one contributing mechanism by which antenatal glucocorticoid therapy improves pulmonary parenchymal immaturity, including surfactant.

Abnormalities, Drug-Induced↗

PubMed1999

Management of ocular perforations resulting from peribulbar anaesthesia.

PURPOSE: To analyze the clinical presentation and outcome of treatment for globe perforation secondary to peri-bulbar anaesthesia. METHODS: Eight patients (3 females and 5 males) aged 66-84 years were included in the study. Ocular perforations were suspected in 3 cases before or during surgery, in 4 cases diagnosis was established within one week and in one case at 3 weeks. Three patients underwent indirect argon laser photocoagulation to seal the retinal break, one patient had cryotherapy, 3 patients underwent a pars plana vitrectomy with fluid gas exchange and endo-laser; and one patient refused any further treatment. RESULTS: The final visual acuity after a mean follow up of 14 months was better than 6/9 in 2 patients, between 6/9-6/12 in 4 patients, and perception of light in 2 patients. CONCLUSION: If diagnosed early and treated adequately, a majority of patients with globe perforation during periocular anaesthetic could be saved.

Aged↗

PubMed1998

Mediastinal lipoma in children.

Mediastinal masses are uncommonly encountered in childhood. These tumours are often interesting because of the varied mediastinal tissues from which they arise. Despite extensive investigations, a large percentage of these lesions remain undiagnosed prior to operation. We describe two rare cases of mediastinal lipoma. The first was incidentally diagnosed whereas the second patient presented with respiratory symptoms. The definitive diagnosis was established at surgery, which was curative.

Child↗

PubMed1998

Active collagen synthesis in infantile hypertrophic pyloric stenosis.

M-57 antibody, which is capable of distinguishing newly-synthesized type I procollagen from fully-processed, mature collagen, was used to examine the expression of collagen synthesis in hypertrophic pyloric muscle from patients with infantile hypertrophic pyloric stenosis (IHPS). Seven specimens from IHPS patients were removed at the time of operation; age-matched normal pyloric tissue of 5 post-mortem cases was obtained as controls. Immunohistochemistry was performed using antibody of the amino-terminal end of the procollagen type I propeptide (M-57). Newly-synthesized procollagen (M-57) was strongly detected in both the connective tissue septa between circular muscle bundles, and among the circular-muscle fibers in patients with IHPS. No M-57 staining was observed among the circular-muscle fibers in controls. Our findings show that the hypertrophic circular muscle in IHPS is actively synthesizing collagen, and this may be responsible for the characteristic "firm" nature of the pyloric tumor.

Antibodies, Monoclonal↗

PubMed1998

Pathogenesis of infantile hypertrophic pyloric stenosis: recent progress.

Although infantile hypertrophic pyloric stenosis (IHPS) is the most common condition requiring surgery in the first few months of life, its pathogenesis is not fully understood. Reviews of the recent progress in the pathogenesis of IHPS show: (1) there is increasing evidence to suggest that smooth-muscle cells in IHPS are not properly innervated; (2) because non-adrenergic, non-cholinergic nerves are mediators of smooth-muscle relaxation, it is likely that the absence of these nerves in pyloric muscle is the cause of excessively contracted hypertrophic circular pyloric muscle; (3) there are abnormal amounts of extracellular matrix proteins in hypertrophic pyloric muscle. Circular muscle cells in IHPS are actively synthesizing collagen, and this may be responsible for the characteristic "firm" nature of the pyloric tumor; and (4) the increased expression of insulin-like growth factor-I, transforming growth factor-beta 1, and platelet-derived growth factor-BB and their receptors in hypertrophic pyloric muscle suggests that increased local synthesis of growth factors may play an important role in smooth-muscle hypertrophy in IHPS.

Extracellular Matrix Proteins↗

PubMed1998

Increased insulin-like growth factor-I mRNA expression in pyloric muscle in infantile hypertrophic pyloric stenosis.

The etiology of infantile hypertrophic pyloric stenosis (IHPS) is unknown. Insulin-like growth factor-I (IGF-I) is a polypeptide hormone that elicits various biological activities (cellular growth, replication, and differentiation) by binding to its receptors. IGF-I has been suggested to play an important role in both gastrointestinal (GI) maturation and smooth-muscle-cell (SMC) hypertrophy. Full-thickness muscle biopsy specimens were obtained from 8 IHPS patients (age range 14-64 days, mean 28.1 days) at pyloromyotomy and from 8 age-matched controls (15-60 days, mean 33.8 days) without GI disease at autopsy. In-situ hybridization was performed using an IGF-I-specific and digoxigenin (DIG)-labeled oligonucleotide probe and visualized by nitroblue tetrazolium staining. In normal controls, IGF-I mRNA expression was absent or weak in both circular and longitudinal smooth-muscle layers of pyloric muscle. In contrast, the pyloric muscle in IHPS patients demonstrated strong IGF-I mRNA expression in the circular smooth-muscle layer and moderate expression in the longitudinal smooth-muscle layer. The increase in IGF-I mRNA in pyloric muscle in IHPS suggests that SMCs are actively synthesizing IGF-I, contributing to the development of pyloric muscle hypertrophy.

Female↗

PubMed1998

NADPH-diaphorase histochemical staining of suction rectal biopsies in the diagnosis of Hirschsprung's disease and allied disorders.

The purpose of this study was to investigate the specificity and sensitivity of NADPH-diaphorase (NADPH-D) staining in suction rectal biopsies (SRB) to determine whether it can be used as a diagnostic test for Hirschsprung's disease (HD) and related disorders. We studied SRB material in 80 patients suspected of having such disorders taken at 3, 5, and 7 cm above the pectinate line. Eight-micron sections were stained with hematoxylin and eosin, acetylcholinesterase histochemistry, and NADPH-D histochemistry. Normal biopsy specimens demonstrated strong NADPH-D reactivity in the submucosal ganglia and a large number of NADPH-D-positive fibers in the muscularis mucosae (MM). In contrast, there were no NADPH-D-positive fibers in the MM in patients with HD and hypertrophic nerve trunks stained weakly. Patients with hypoganglionosis (HYPG) demonstrated only a few NADPH-D-positive fibers in the MM and scant submucosal ganglia. Our results show that it is possible to diagnose HD and HYPG in mucosal rectal biopsies containing MM only and stained by NADPH-D histochemistry. As there is no background staining in NADPH-D histochemistry, it is easy to detect NADPH-D-positive fibers. NADPH-D histochemical staining may be an important additional technique for diagnosing HD and related disorders.

Biopsy, Needle↗

PubMed1998

Lack of a docking mechanism for neurotransmitter release in the aganglionic segment of bowel in patients with Hirschsprung's disease.

We examined immunohistochemically the expression and localisation of synapse-associated proteins, syntaxin (SNT) and synaptotagmin (STG) in the entire resected specimens of colon obtained at the time of pull-through operation from 15 patients with Hirschsprung's disease (HD) and 6 age-matched controls. Both antibodies showed a similar pattern of staining. In the normal colon and ganglionic colon from HD, there was strong reactivity in the submucous and myenteric plexuses in addition to staining of nerve fibres in the smooth-muscle layers. In the aganglionic colon, there was an absence or marked decrease in SNT and STG-positive nerve fibres in the smooth-muscle layers and in hypertrophic nerve trunks. Our data indicate that important proteins necessary for the docking of synaptic vesicles at the presynaptic plasma membrane are lost in fibres innervating the smooth muscle of HD and suggest that abnormal neurotransmission may have a role in the maintenance of muscle spasticity.

Calcium-Binding Proteins↗

PubMed1998

Upregulated tumor necrosis factor-alpha gene expression in the hypoplastic lung in patients with congenital diaphragmatic hernia.

Recent studies using animal models of congenital diaphragmatic hernia (CDH) have reported a reduction in both surfactant (SF) phospholipids and proteins in CDH lungs compared to controls, resulting in biophysical and physiologic impairment of SF function in the hypoplastic CDH lung. Furthermore, SF replacement has been shown to improve physiological function in CDH lungs. Tumor necrosis factor-alpha (TNF-alpha) is a polypeptide whose overproduction has been implicated in the pathogenesis of a number of pathological conditions, such as neonatal and adult respiratory distress syndrome. TNF-alpha has been shown to selectively inhibit the de-novo synthesis of SF phospholipid components in type II pneumocytes. It has been demonstrated that TNF-alpha is synthesized locally in lung and functions in an autocrine/paracrine mode. The aim of this study was to investigate TNF-alpha messenger RNA (mRNA) expression in hypoplastic CDH lung using in-situ hybridization histochemistry, to determine the molecular basis of the SF deficiency in the hypoplastic CDH lung. Lung-tissue samples were obtained at autopsy from 7 full-term newborns (age range: 1-21 days) with CDH and 4 stillborns with CDH. Normal lung tissue from eight infants with sudden infant death syndrome (age range: 5-30 days) acted as controls. In-situ hybridization was performed using TNF-alpha specific and digoxigenin-labeled oligonucleotide probe and visualized by nitroblue tetrazolium staining. In control lung tissue, mRNA expression of TNF-alpha was absent or weak in type II pneumocytes and alveolar macrophages. In contrast, mRNA expression of TNF-alpha was markedly increased in both type II pneumocytes and alveolar macrophages in hypoplastic CDH lung. Our findings of up-regulated TNF-alpha gene expression in CDH lung suggest that the SF deficiency observed in hypoplastic CDH lung may be the result of increased local production of TNF-alpha.

Case-Control Studies↗

PubMed1998

Comparison of the pulmonary vasculature in newborns and stillborns with congenital diaphragmatic hernia.

The purpose of this study was to compare structural changes in the pulmonary vasculature in newborns with congenital diaphragmatic hernia (CDH) complicated by persistent pulmonary hypertension (PPH) and stillborns with CDH. Victorian blue van Gieson (VVG) staining and immunostaining with anti-alpha smooth-muscle actin (ASMA) was performed on lung tissue obtained at autopsy from 23 newborns with CDH complicated by PPH, 7 stillborns with CDH, and 11 age-matched controls with sudden infant death syndrome (SIDS). The degrees of adventitial and medial thickness and area were measured in pulmonary arteries with an external diameter (ED) of <75 micrometers, 75-100 micrometers, 100-150 micrometers, 150-250 micrometers, 250-500 micrometers, and >500 micrometers by image analyzer and compared statistically. The degrees of adventitial and medial thickness and area were measured in pulmonary veins with an ED of <100 micrometers, 100-200 micrometers, and >200 micrometers by image analyzer and compared statistically. In order to determine whether the characteristic structural changes were size-related, each was related to ED. There was a significant increase in adventitial thickness and area in arteries of all sizes in both newborns and stillborns with CDH compared to SIDS patients (P < 0. 05). The degree of medial thickness in newborns and stillborns with CDH was significantly increased compared to SIDS patients (P < 0.01). The degree of medial area was significantly increased for arteries with ED less than 100 micrometers (P < 0.05) in newborns and stillborns with CDH compared with SIDS patients. There was a significant increase in adventitial thickness and area in veins of all sizes in newborns with CDH compared to stillborns with CDH and SIDS (P < 0. 05). The degree of adventitial thickness and area of pulmonary veins were similar in stillborns with CDH and SIDS. There were no significant differences in medial thickness of veins between the three groups. The presence of abnormally thick-walled pulmonary arteries in stillborns with CDH suggests that the intrapulmonary arteries in CDH may become excessively muscularized during fetal life, becoming unable to adapt normally at birth. The absence of structural changes in pulmonary veins in stillborns with CDH suggests that the pulmonary venous changes observed in newborns with CDH complicated by PPH occur after birth as a result of increases in transvascular pressure or a response to release of peptide growth factors.

Case-Control Studies↗