Preventive and therapeutic effects of relaxin on bisphosphonate related osteonecrosis of the jaw: an experimental study in rats / Efeitos preventivos e terapêuticos da relaxina na osteonecrose da mandíbula relacionada a bisfosfonatos: um estudo experimental em ratos

Objective: Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a challenging complication of chronic bisphosphonate (BP) use. The hormone relaxin is able to induce the multistep differentiation process of human osteoclastogenesis, exhibits antifibrotic and anti-inflammatory actions, and promotes vasodilatation, wound healing, and angiogenesis. The present study aimed to evaluate the effects of relaxin in the prevention and management of BRONJ. Material and Methods: Thirty-six male Sprague Dawley rats were randomly divided into four groups. Rats in group 1 (n = 10) received relaxin and BP simultaneously for 12 weeks. Rats in group 2 (n = 10) received injections of BP for 12 weeks, followed by relaxin for another 12 weeks. Rats in group 3 (n = 10) received only BP injections, and those in group 4 (control, n = 6) received only saline. Necrosis and inflammation in the rats' mandibles were evaluated as indicators of BRONJ. Results: Necrosis and inflammation were not detected in group 1 (BP + relaxin). In group 3 (BP only), incidence rates of necrosis and inflammation were 90% and 60%, respectively. Conclusions: Our findings suggest that relaxin may be potently effective in preventing BRONJ and have some benefit in the treatment of existing BRONJ (AU)

Objetivo: A osteonecrose da mandíbula relacionada ao bisfosfonato (BRONJ) é uma desafiadora complicação do uso crônico de bisfosfonato (BP). O hormônio relaxina é capaz de induzir o processo múltiplo de diferenciação da osteoclastogênese humana, exibe ações anti-fibróticas e anti-inflamatórias e promove vasodilatação, cicatrização de feridas e angiogênese. O presente estudo teve como objetivo avaliar os efeitos da relaxina na prevenção e tratamento do BRONJ. Material e Métodos: Trinta e seis ratos Sprague Dawley machos foram divididos aleatoriamente em quatro grupos. Os ratos do grupo 1 (n = 10) receberam relaxina e BP simultaneamente por 12 semanas. Os ratos do grupo 2 (n = 10) receberam injeções de BP por 12 semanas, seguidos de relaxina por mais 12 semanas. Os ratos do grupo 3 (n = 10) receberam apenas injeções de BP e os do grupo 4 (controle, n = 6) receberam apenas solução salina. Necrose e inflamação nas mandíbulas dos ratos foram avaliadas como indicadores de BRONJ. Resultados: Necrose e inflamação não foram detectadas no grupo 1 (BP + relaxina). No grupo 3 (somente BP), as taxas de incidência de necrose e inflamação foram de 90% e 60%, respectivamente. Conclusões: Nossos resultados sugerem que a relaxina pode ser potentemente eficaz na prevenção do BRONJ e ter algum benefício no tratamento do BRONJ existente.(AU)

Effect of Relaxin Expression from an Alginate Gel-Encapsulated Adenovirus on Scar Remodeling in a Pig Model

PURPOSE: Relaxin (RLX) is a transforming growth factor-β1 (TGF-β1) antagonist that is believed to function as a potent collagen re-arranger and a major suppressor of extracellular matrix components. Adenoviruses (Ads) are accepted vectors for cancer gene therapy. However, repeated treatments of Ad are limited by short-term biological activity in vivo. The efficacy of sustained RLX expression to scar remodeling was assessed using an injectable alginate gel-matrix system. MATERIALS AND METHODS: Pig scar tissue was treated with relaxin-expressing Ad loaded in alginate gel (gel/Ad-RLX). Surface areas, color, and pliability of scars were compared, and various factors influencing scar formation and collagen arrangement were analyzed. RESULTS: Gel/Ad-RLX decreased scar size, color index, and pliability. Immunohistochemistry showed decreased levels of major extracellular matrix proteins in the gel/Ad-RLX-treated group. Furthermore, treatment with gel/Ad-RLX reduced expression of tissue inhibitor of metalloproteinase-1 and alpha-smooth muscle actin and markedly increased expression of matrix metalloproteinase-1 in pig scar tissues. Gel/Ad-RLX also significantly downregulated TGF-β1 and upregulated TGF-β3 mRNAs in pig scar tissues. CONCLUSION: These results support a prominent role for RLX in scar remodeling and suggest that gel/Ad-RLX may have therapeutic effects on scar formation.

Relaxin Modulates the Expression of MMPs and TIMPs in Fibroblasts of Patients with Carpal Tunnel Syndrome

PURPOSE: The aim of this study was to investigate the anti-fibrotic effect of relaxin in subsynovial fibroblasts activated by transforming growth factor beta (TGF-β). MATERIALS AND METHODS: To test the anti-fibrotic effect of an adenovirus-relaxin construct (Ad-RLN) on subsynovial fibroblasts in vitro, cells from subsynovial connective tissue of patients with carpal tunnel syndrome were activated with TGF-β1 and exposed to Ad-RLN (as a therapeutic gene) or adenovirus-lacZ construct (as a marker gene) for four hours. Subsynovial fibroblast cultures without adenoviral exposure served as controls. RESULTS: We observed induction of gene expressions of collagen I, III and IV, as well as the abatement of alpha-smooth muscle actin (a-SMA) synthesis, Smad2 phosphorylation, and fibronectin at the protein level, in comparison to controls. In addition, protein expressions of matrix metalloproteinase (MMP) I was significantly induced, whereas the protein expressions of tissue inhibitor of metalloproteinases (TIMP) I and IV were reduced due to relaxin expression. CONCLUSION: RLN prevents excessive synthesis of extracellular matrix by reducing the expressions of its components, such as fibronectin, a-SMA, and phosphorylated Smad2, by increasing the expression of MMPs; and by decreasing the expression of TIMPs.

Effect of Relaxin Expressing Adenovirus on Scar Remodeling: A Preliminary Study

BACKGROUND: Relaxin is a transforming growth factor β1 antagonist. To determine the effects of relaxin on scar reduction, we investigated the scar remodeling process by injecting relaxin-expressing adenoviruses using a pig scar model. METHODS: Scars with full thickness were generated on the backs of Yorkshire pigs. Scars were divided into two groups (relaxin [RLX] and Control). Adenoviruses were injected into the RLX (expressing relaxin) and Control (not expressing relaxin) groups. Changes in the surface areas, color index and pliability of scars were compared. RESULTS: Fifty days after treatment, the surface areas of scars decreased, the color of scars was normalized, and the pliability of scars increased in RLX group. CONCLUSION: Relaxin-expressing adenoviruses improved the surface area, color, and pliability of scars. The mechanism of therapeutic effects on scar formation should be further investigated.

Mechanism of Ligamentum Flavum Hypertrophy / 대한정형외과연구학회지

Ligamentum flavum (LF) is yellowish ligament tissue connecting the lamina of adjacent vertebra. Degenerative changes in the spine cause the hypertrophy of LF and facet joint and disc bulging and herniation. These changes results in a narrowing of the spinal canal. Neural decompression surgery by removing the hypertrophied lamina, LF and disc pathologies has been considered as successful treatment method in lumbar spinal stenosis. This surgery has showed relatively satisfactory clinical results and has increased life-expectancy in elderly patients. However, issues about post spinal surgery syndrome and re-stenosis after the surgery also have been reported. Because LF is one of the main mechanisms of spinal stenosis, accurate understanding about pathologic mechanism on the LF hypertrophy may suggest alternative treatment methods such as medical treatment or less invasive treatment than surgical decompression can be considered. Hypertrophy of the ligamentum flavum is generated from increase of collagen synthesis, fibroblast proliferation, and fibrosis caused by 1) the expression of growth factors (TGF-beta1 etc.) stimulated by the repeated mechanical tension, 2) inflammatory cytokines from spinal facet joint structure and LF 3) delayed cell death, and 4) inflammatory cytokine from hypertrophied and degenerated LF itself. After the middle ages, gradual and partial inhibition of LF hypertrophy can be expected by administration NSAIDs or selective cyclo-oxygenase-2 inhibitors because these drugs may cause reduction of the increased cytokines. Also, relaxin can be another new treatment material for spinal stenosis by the mechanism of melting hypertrophied LF and reducing synthesis of collagen.

Relaxin inhibit cardiac fibrosis induced by phorbol 12-myristate 13-acetate / 生物医学与环境科学（英文）

Relaxin is known to inhibit cardiac fibrosis. However, it is unclear whether relaxin could regulate the effects of Phorbol 12-myristate 13-acetate (PMA, PKC activator) on cardiac fibrosis. So the influence of relaxin on the cell proliferation and collagen expression induced by PMA in cultured cardiac fibroblasts was studied. It showed that PMA significantly increased cardiac fibroblasts proliferation, Type I pro-collagen protein expression, Type I pro-collagen mRNA expression, and rhRLX absolutely significantly decreased PMA induced effects on cardiac fibroblasts proliferation and Type I pro-collagen expressions, indicating that relaxin could inhibit cardiac fibrosis induced by PMA.

Cultura de células da granulosa humanas com fenótipo de fase folicular: influência do sistema quimicamente definido na morfologia, ultraestrutura, secreção de esteroides e relaxina / Culture of human granulosa cells with follicular phase phenotype:influence of chemically defined system on morphology, ultrastructure,secretion of steroids and relaxin

Está bem descrito na literatura o padrão de cultivo de células da granulosa (CG) humanas que perpetua a luteinização, simulando a fase lútea do ciclo. Nesse sistema, há redução na secreção de estradiol (E2) e aumento na síntese de progesterona (P4) e relaxina (RLN). Objetivamos padronizar um sistema de cultura livre de soro, com o intuito de reverter o processo de luteinização de CG obtidas em ciclos de fertilização in vitro (FIV), pré-luteinizadas pela gonadotrofina coriônica humana (hCG), para aplicação na maturação in vitro de folículos ovarianos pré-antrais. Foi feito estudo experimental com GC obtidas de 10 mulheres em tratamento de reprodução assistida. As CG foram cultivadas em α-MEM contendo IGF-I, ITS, androstenediona, PVP-40 (meio quimicamente definido) ou TCM-199 contendo FSH/soro. Após 48, 96 e 144 horas, foram avaliados: morfologia das culturas, produção de E2, P4 (Quimioluminescência/Immulite), RLN (Elisa) e ultraestrutura (Microscopia Eletrônica). Os dados foram analisados por Anova e regressão linear com efeitos mistos (SAS versão 9.0). Células cultivadas em α-MEM apresentam alta capacidade estrogênica e padrão de produção hormonal característico da fase folicular, mantendo características morfológicas/ultraestruturais semelhantes a células in vivo. No sistema de cultura padronizado, as CG não completam in vitro o processo de luteinização deflagrado pela hCG, assumindo fenótipo de fase folicular.

It is well described in the literature the granulosa cells (GC) culture pattern that perpetuates human luteinizing simulating the luteal phase of the cycle. In this system, there is a reduction in the secretion of estradiol (E2) and increased synthesis of progesterone (P4) and relaxin (RLN). We aim to standardize a serum-free culture system, in order to reverse the luteinization process of GC obtained in IVF cycles, pre-luteinized by hCG, for use in in vitro maturation of preantral ovarian follicles. An experimental study was conducted with GC obtained from10 women undergoing treatment for assisted reproduction. The GC were cultured in α-MEM containing IGF-I, STI, androstenedione, PVP-40 (chemically defined medium) or TCM-199 containing FSH/serum. After 48, 96 and 144 h were analyzed: culture morphology, concentrations of E2, P4 (Chemioluminescence/Immulite), and RLN (Elisa), and ultrastructure (ElectronMicroscopy). Data were analyzed by Anova and linear mixed-effects regression (SAS version9.0). Cells cultured in α-MEM present estrogenic capacity and pattern of hormone production characteristic of the follicular phase, maintaining morphological/ultrastructural features similar that in vivo cell. In standard culture system, the CG not completes in vitro luteinization process triggered by hCG, assuming follicular phase phenotype.

Métodos farmacológicos de indução do trabalho de parto: qual o melhor? / Pharmacological methods for induction of labor: what's the best?

A indução do parto consiste em estimular artificialmente as contrações uterinas coordenadas e efetivas antes de seu início espontâneo, levando ao desencadeamento do trabalho de parto em mulheres que ultrapassaram a 22ª semana de gravidez. A antecipação do parto pode ser necessária em diversas situações obstétricas, como gestação prolongada, diabetes, ruptura prematura das membranas e pré-eclâmpsia. Estima-se que mais de 15% de todas as gestantes apresentem alguma indicação de indução do parto. Vários métodos de indução do parto são propostos, tanto naturais como artificiais e, dentre estes, os métodos farmacológicos merecem especial destaque. Realizou-se uma revisão da literatura baseada nos melhores níveis de evidências e considerando os graus de recomendação. De acordo com a literatura, a utilização de estrogênio, propranolol, relaxina, mefepristone e hialuronidade não deve ser estimulada por não existirem evidências suficientes para a sua recomendação. O seu uso, portanto, deve ser limitado a protocolos de pesquisas. Ocitocina é um método de indução efetivo que pode ser usado em pacientes com ruptura das membranas amnióticas. Prostaglandinas (PG) e misoprostol (um éster sintético da PGE1) são efetivos para a indução do parto independentemente da integridade das membranas. Prostaglandinas devem ser administradas preferencialmente por via vaginal. Habitualmente, o misoprostol é preferido devido a questões práticas, como o baixo custo e a facilidade de administração e estocagem. Doses baixas de misoprostol devem ser utilizadas e a atualmente recomendada é de 25 g a cada 4 ou 6 horas. Tanto a via oral como a via vaginal podem ser utilizadas.

Induction of labor consists of stimulation of effective and coordinated uterine contractions before their spontaneous onset for the purpose of bring on labor in women who have surpassed the 22nd week of pregnancy. In several obstetrical situations, such as prolonged pregnancy, diabetes, premature rupture of membranes and preeclampsia, anticipation of labor and delivery may be necessary. It is estimated that more than 15% of all pregnant women eventually present any indication for induction of labor. Several natural and artificial methods for induction are proposed. Among them, pharmacological methods are the most relevant. A literature review was carried out based on the highest levels of evidence and on the grade of recommendations. According to the literature, the use of estrogens, relaxin, mifepristone and hyaluronidade should not be stimulated because there are not enough evidences for their recommendation, so their utilization should be limited to research protocols. Oxytocin is an effective method for induction of labor that may be used in patients with ruptured membranes. On the other hand, prostaglandins and misoprostol (a prostaglandin E1 analog) are effective for induction of labor independently on the membrane integrity. Vaginal administration should be preferred for prostaglandins. Misoprostol is habitually preferred due to practical questions, such as low cost and facility for storage and administration. Low doses of misoprostol should be used, and the currently recommended dose is 25 g every four or six hours. Both vaginal and oral routes of administration can be used.

Relaxina en suero de mujeres embarazadas, a nivel del mar y en la altura / Relaxin in serum of pregnant women, at sea level as high altitude

Se estudió los niveles en suero de relaxina en el 3°, 4° y 5° mes de gestación, así como los niveles en suero de mujeres posparto y en recién nacidos dentro de las primeras 24 horas, tanto a nivel del mar como de altura. El estudio se realizó en 24 mujeres embarazadas (18 a 32 años), en el tercer, cuarto o quinto mes de gestación natural de Cerro de Pasco, (4200 msnm) y 20 mujeres embarazadas de la misma edad y estado gestacional nativas del nivel del mar (Lima, 150 m). Asimismo, se estudiaron los niveles en suero de relaxina en 09 mujeres y sus 09 recién nacidos dentro de las siguientes 24 horas después del parto para el grupo de altura, y 20 mujeres con sus 20 recién nacidos dentro de las siguientes 24 horas, a nivel del mar. La relaxina fue medida por RIA (Radioinmunoanálisis), con el uso de kits de los Laboratorios Immunodiagnostik (Alemania) marcada con 1-125 y con el uso del equipo Contador de centelleo gamma del laboratorio del Instituto de Investigaciones Clínicas de la Universidad Nacional Mayor de San Marcos. Los resultados nos indican que no hay diferencia significativa entre los niveles hormonales de relaxina en el 3°, 4° Y 5° mes de gestación en las mujeres del nivel del mar y altura; existiendo una gradual disminución de los niveles en suero del 3° al 5° mes tanto a nivel del mar como en la altura. En las mujeres post parto, los niveles de relaxina en suero fueron significativamente menores en las mujeres de altura con respecto a las del nivel del mar. El radio relaxina madre/relaxina hijo es igual en la altura como en el nivel del mar.

We studied serum levels of relaxin in the 3th, 4th and 5th month of pregnancy, women postpartum and newborns within the first 24 hours both at sea level as high altitude. The study was performed in 24 pregnant women (18 to 32 years) in the third, fourth or fifth month of gestation in (Cerro de Pasco, 4300 m) and 20 pregnant women of the same gestational age at sea level (Lima, 150 m). Also, we studied the serum levels of relaxin in 09 women and 09 newborns within 24 hours postpartum at the high altítude group and 20 women and 20 newborns within 24 hours at sea level. Relaxin was measured by RIA (Radioimmunoassay), using kits Immunodiagnostic Laboratories (Germany) labeled with 1-125 and using the gamma scintillation counter computer in lab of the Institute of Clinical Research at the National University of San Marcos. The results indicate no significant difference between the hormone levels of relaxin in the 3 th, 4 th and 5 th month of pregnancy in women at high altitude and sea level, showing a gradual decrease in serum levels of 3 th to 5 th month both at sea level and at altitude. In postpartum women, levels of relaxin in serum were significantly lower in women at high altitude with regard to sea level. The radio mother relaxin / child relaxin is the same at high altitude as the sea level.

Effect of Relaxin Expressing Adenovirus for Rat Skin Flap Viability

PURPOSE: Of various effects of relaxin, we assumed that anti-fibrotic effects, neovascularization effects and vasodilatation effects of relaxin might enhance the survival rate of skin flap. In the current study, we used adenovirus expressing relaxin genes to examine whether these genes could enhance the survival rate of a skin flap. METHODS: A total of 30 Sprangue-Dawley rats were divided into three groups: RLX group (10; relaxin virus injected group), CTR group (10; no gene coded virus injection group), and PBS group (10; PBS injected group). Each group was intradermally injected with the virus (107 PFU) and PBS 48 hours before and immediately before the flap elevation. A distally based flap 3 x 9 cm in size was elevated on the dorsal aspect of each rat. Following this, a flap was placed in the original location and then sutured using a #4-0 Nylon. A surviving area of the flap was measured and then compared on postoperative days 3, 7 and 10. Using a laser Doppler, the amount of blood flow was measured. On postoperative day 10, tissues were harvested for histologic examination and the number of blood vessels was counted. RESULTS: There was a significant increase in the area of the flap survival in the RLX group on postoperative days 3 and 7. The Doppler measurement also showed significantly increased blood flow immediately after the operation and on postoperative days 7 and 10. The number of blood vessels was significantly greater in the RLX group in the tissue harvested on postoperative day 10. The VEGF concentration was significantly higher in the RLX group than others in the tissues harvested on postoperative day 10. CONCLUSION: Following an analysis of the effects of relaxin-secreting adenovirus on the survival of a flap, the surviving area of the flap and the blood flow also increased. A histopathology also showed an increase in the number of blood vessels and the concentration of VEGF.

Effect of porcine relaxin on NO production of human microvascular endothelial cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe effect of porcine relaxin(pRLX) on NO production of human microvascular endothelial cells(HMVECs) and discuss its possible mechanism.</p><p><b>METHODS</b>iNOS and cNOS expression of HMVECs with or without pRLX were detected using western blotting. NO production of HMVECs with pRLX at different concentration or different time were determined by method of Griess. NO production of pRLX of HMVECs plus Non-selective NOS inhibitor NG-monomethyl-L-arginine(L-NMMA), selective iNOS inhibitor aminoguanidine(AG) or nuclear factors-kappaB (NF-kappaB) inhibitor pyrrolidine dithiocarbamate(PDTC) were also analysed.</p><p><b>RESULTS</b>pRLX promoted iNOS protein expression of HMVECs, but not cNOS protein expression. NO production of HMVECs was promoted by pRLX on concentration-dependent pattern instead of time-dependent one. AG, L-NMMA and PDTC were showed to block the effect of pRLX on NO production of HMVECs.</p><p><b>CONCLUSION</b>pRLX promote iNOS expression and NO production of HMVECs.</p>

Métodos para indução do parto / Methods for labor induction

O interesse da obstetrícia moderna pela indução do parto é demonstrado pela grande quantidade de artigos científicos publicados nos últimos anos. Os avanços da medicina em geral e da obstetrícia em particular têm permitido que mais gestações de risco evoluam até o termo ou próximo dele, com indicação materna ou fetal de interrupção da gestação antes do desencadeamento do trabalho de parto espontâneo. Isso coloca o obstetra na situação entre a escolha da cesárea ou da indução do parto. Para que o obstetra faça a escolha pela indução do parto e desta forma colabore com a diminuição da incidência de cesárea, é necessário que haja método acessível, barato, seguro, efetivo, de fácil utilização e de boa aceitabilidade. Embora exista grande quantidade de métodos de indução do parto relatados na literatura médica, sabe-se que ainda não há método ideal. No entanto, dentre eles, dois se destacam. O primeiro é a ocitocina, que possui as vantagens de promover contrações uterinas fisiológicas de trabalho de parto e com possibilidade de reverter os quadros de aumento da contratilidade uterina com a sua suspensão. O outro método é o misoprostol, o mais utilizado na atualidade, que amadurece o colo uterino e provoca contrações uterinas de trabalho de parto. No entanto, em relação ao misoprostol ainda existem controvérsias sobre sua dose e via ideal e segurança

Three Cases of Separation of Symphysis Pubis During Vaginal Delivery / 대한주산의학회잡지

Separation of symphysis pubis during vaginal delivery is rare condition with incidence ranging from 1/500 to 1/30000 deliveries. The injury is caused by fetal head exerting pressure on pelvic ligaments that have been relaxed by progesterone and relaxin. The separation might be associated with considerable pain, swelling and tenderness over the pubic area. Diagnosis is based on clinical findings and X-ray findings. The condition is treated conservatively with bed rest, analgesics and physical therapy. Prognosis is exellent. We experienced 3cases of separation of symphysis pubis during vaginal delivery and report these cases with a brief review of literature.

Mecanismo endrocinologicos envolvidos no parto prematuro / Endocrinological mechanism involved in the premature birth

O objetivo deste estudo e apresentar os mecanismos endocrinologicos no parto prematuro. Os principais hormonios conhecidos sao...

Traumatic Separation of the Symphysis Pubis / 대한정형외과학회잡지

Symphyseal injury is increasing in number together with today's speed of development of car industry in Korea. However, this injury is not common in practice. Some authors reported that symphyseal injury is only 4 to 6% of all pelvic fractures. Symphysis pubis has characteristicsl anatomy to maintain mechanical integrity of the pe1vis with circumferential ligament. The pelvis is a ring structure with strong ligaments. This support include the symphysis pubis, the anterior and posterior sacroiliac ligaments, and the strong sacrotuberous ligaments. According to Peltier(1964), when symphysis is separated more than 1.0cm, pubic instability will be developed. However, Wild(1982) reported that pelvic instability develops when separation of the symphysis exceeds more than 2.5cm. Tile(1984) reported that anterior pubic rami acts as a strut to prevent anterior collapse of the pelvic ring during weight bearing. However, in the presence of intact posterior structures, it gives little effect on pelvic stability. In addition to trauma, pelvic instability develops congenitally or by pregnancy. During pregnancy, pregnancy-related hormones relsx the ligameritous stuctures of the pelvic girdle. In most instances, the major pelvic ring returns to normal when the effect of the relaxin hormones disappear. However, in rare instances, a major symphysis disruption may continuously persist. To evaluste the trauma-induced separation of the symphysis pubis, we analyzed the 19 cases with 15 months follow-up on an average, who were treated at the Orthopaedic Department, Kang-Nam St. Marys Hospital, from June 1981 to June 1986. The results were as follows 1. Among 19 cases, 9 cases(47.4%) were male, 10 cases(52.6%) were female. And average age of the patients was 30.2 years. 2. The main cause of the fracture was traffic accident in 18 out of 19 cases. 3. In cases of symphyseal separation more than 3.4cm, fracture-separation of both sacroiliac joint was certainly occured. However, in cases with separation more than 2.2cm, unilalateral fracture-dislocation of sacroiliac joint occurred. 4. Open reduction and interal fixatiopn including external fixation was performed in 9 og cases. As an indication of surgery, separation of the symphysis, which exceeds more than 2.2cm and which associated (1) with sacroiliac fracture-dislocation, (2) failed conservative treatment, and (3) when simultaneously emergency urological operation is indicated.