POTENTIAL DIAGNOSTIC BIOMARKERS FOR HUMAN MESENCHYMAL TUMORS, ESPECIALLY LMP2/Β1I AND CYCLIN E1/MIB1 DIFFERENTIAL EXPRESSION: PRUM-IBIO STUDY.

Most mesenchymal tumors found in the uterine corpus are benign tumors; however, uterine leiomyosarcoma is a malignant tumor with unknown risk factors that repeatedly recurs and metastasizes. In some cases, the histopathologic findings of uterine leiomyoma and uterine leiomyosarcoma are similar and surgical pathological diagnosis using excised tissue samples is difficult. It is necessary to analyze the risk factors for human uterine leiomyosarcoma and establish diagnostic biomarkers and treatments. Female mice deficient in the proteasome subunit low molecular mass peptide 2 (LMP2)/ß1i develop uterine leiomyosarcoma spontaneously. MATERIAL AND METHODS: Out of 334 patients with suspected uterine mesenchymal tumors, patients diagnosed with smooth muscle tumors of the uterus were selected from the pathological file. To investigate the expression status of biomarker candidate factors, immunohistochemical staining was performed with antibodies of biomarker candidate factors on thin-cut slides of human uterine leiomyosarcoma, uterine leiomyoma, and other uterine mesenchymal tumors. RESULTS AND DISCUSSION: In human uterine leiomyosarcoma, there was a loss of LMP2/ß1i expression and enhanced cyclin E1 and Ki-67/MIB1 expression. In human uterine leiomyomas and normal uterine smooth muscle layers, enhanced LMP2/ß1i expression and the disappearance of the expression of E1 and Ki-67/MIB1 were noted. The pattern of expression of each factor in other uterine mesenchymal tumors was different from that of uterine leiomyosarcoma. CONCLUSIONS: LMP2/ß1i, cyclin E1, and Ki-67/MIB1 may be candidate factors for biomarkers of human uterine leiomyosarcoma. Further large-cohort clinical trials should be conducted to establish treatments and diagnostics for uterine mesenchymal tumors.

POST-COVID-19 INFLAMMATORY RHEUMATOID ARTHRITIS REMISSION.

People infected with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) are at a higher risk of developing autoimmune inflammatory rheumatic disease. However, clinical studies have shown that, unlike bacterial infections, inflammatory rheumatoid arthritis is rarely triggered by viral infections. Generally, adult females have a higher incidence of rheumatoid arthritis compared to males (a female/male ratio of approximately 3:1). The secretion of female hormones is presumed to be deeply involved in the onset of rheumatoid arthritis. Furthermore, there is a definitive role of genetic factors in rheumatoid arthritis. Typically, rheumatoid arthritis is treated with steroids and antibody drugs, such as anti-tumor necrosis factor-α (TNF-α) antibodies and anti-interleukin-6 (IL-6) antibodies; however, although the symptoms of autoimmune diseases are alleviated by these drugs, the underlying pathology cannot be completely cured. Meanwhile, immunosuppressive treatment with steroids is effective against inflammatory rheumatoid arthritis associated with coronavirus disease (COVID-19). Therefore, the pathogenesis, symptoms, and pathological findings of inflammatory rheumatoid arthritis associated with COVID-19 are presumably different from those of autoimmune rheumatoid arthritis. Since COVID-19-related autoimmune-like diseases, such as COVID-19-related inflammatory rheumatoid arthritis, have pathological conditions that are different from inherited autoimmune diseases, it is possible to establish treatments that aim at remission. Further pathological analyses of patients with post-COVID-19 inflammatory rheumatoid arthritis are essential to the development of treatments for this type of arthritis.

POTENTIAL LIFE PROGNOSTIC MARKER FOR MESENCHYMAL TUMOR RESEMBLING UTERINE LEIOMYOSARCOMA.

Benign uterine leiomyoma (U.LMA) and malignant uterine leiomyosarcoma (U.LMS), both uterine mesenchymal tumors, are distinguished by the number of cells exhibiting mitotic activity. However, uterine mesenchymal tumors contain tumor cells with various cell morphologies; therefore, making a diagnosis, including differentiating between benign and malignant tumors, is difficult. For example, cotyledonoid dissecting leiomyoma (CDL) or uterine smooth muscle tumors of uncertain malignant potential (STUMPs) are a group of uterine mesenchymal tumors for which a differential diagnosis is challenging. To date, a standardized classification system for uterine mesenchymal tumors has not yet been established. Furthermore, definitive preoperative imaging techniques or hematological examinations for the potential inclusion of CDL or STUMP in the differential diagnosis have not been defined. Several clinical studies have reported that there is no correlation between biomarker expression and mitotic rate or tumor recurrence. The immunohistochemical biomarkers reported so far cannot effectively help determine the malignant potential of CDL or STUMPs in patients who wish to become pregnant in the future. The establishment of gene expression profiles or detection of pathogenic variants by using next-generation molecular techniques can facilitate disease prediction, diagnosis, treatment, and prognosis. We examined the oncological properties of STUMP in adults using molecular pathological techniques on tissue excised from patients with uterine mesenchymal tumor. In a clinical study conducted by our medical team, the results of gene expression profiling indicated factors that may be associated with malignancy of uterine mesenchymal tumors. We herein describe the problems in diagnosing uterine mesenchymal tumors along with the results of the latest clinical studies. It is expected that the establishment of a diagnostic method targeting the characteristics of mesenchymal tumor cells will lead to the treatment of malignant tumors with a low risk of recurrence and metastasis.

EFFECT OF RBD MUTATIONS IN SPIKE GLYCOPROTEIN OF SARS-COV-2 ON NEUTRALIZING IGG AFFINITY.

Certain mutant strains of SARS-CoV-2 are known to spread widely among humans, including the receptor binding domain (RBD) mutant, Y453F, from farmed minks, and the RBD mutant, N501Y, a mutation common to three major SARS-CoV-2 subvariants (B.1.1.7, B.1.351, and B.1.1.248) and omicron type SARS-CoV-2 BQ.1.1 and XBB.1.16 subvariants. We investigated the characteristics of the RBD mutants, Y453F and N501Y, using three-dimensional structural analysis. We also investigated the effect of Y453F, N501Y or the mutants of RBD of omicron type SARS-CoV-2 BQ.1.1 and XBB.1.16 subvariants on neutralizing antibodies in serum derived from individuals including children (aged 5-11 years) inoculated with mRNA based COVID-19 vaccine (BNT162b2: Pfizer/BioNTech) or COVID-19-positive patients or children (aged 5-11 years) after vaccination with BNT162b2. Our results suggest that SARS-CoV-2 subspecies with the RBD mutations Y453F or N501Y partially escaped detection by 4 neutralizing monoclonal antibodies and 21 neutralizing antibodies in serums derived from COVID-19-positive patients. The significantly low antibody titer of children against Omicron type SARS-CoV-2 BQ.1.1 subvariant and XBB.1.16 subvariant in Japan. Infection with SARS-CoV-2 subspecies that causes serious symptoms in humans may spread globally. In particular, since the antibody titer against the omicron type is low in children (aged 5-11 years) who have been vaccinated with conventional vaccines, therefore it is important for children to receive vaccines specific for the omicron type.

IFN-γ from lymphocytes induces PD-L1 expression and promotes progression of ovarian cancer.

BACKGROUND: PD-L1 (programmed cell death 1 ligand 1) on tumour cells suppresses host immunity through binding to its receptor PD-1 on lymphocytes, and promotes peritoneal dissemination in mouse models of ovarian cancer. However, how PD-L1 expression is regulated in ovarian cancer microenvironment remains unclear. METHODS: The number of CD8-positive lymphocytes and PD-L1 expression in tumour cells was assessed in ovarian cancer clinical samples. PD-L1 expression and tumour progression in mouse models under conditions of altering IFN-γ signals was assessed. RESULTS: The number of CD8-positive cells in cancer stroma was very high in peritoneally disseminated tumours, and was strongly correlated to PD-L1 expression on the tumour cells (P<0.001). In mouse models, depleting IFNGR1 (interferon-γ receptor 1) resulted in lower level of PD-L1 expression in tumour cells, increased the number of tumour-infiltrating CD8-positive lymphocytes, inhibition of peritoneal disseminated tumour growth and longer survival (P=0.02). The injection of IFN-γ into subcutaneous tumours induced PD-L1 expression and promoted tumour growth, and PD-L1 depletion completely abrogated tumour growth caused by IFN-γ injection (P=0.01). CONCLUSIONS: Interferon-γ secreted by CD8-positive lymphocytes upregulates PD-L1 on ovarian cancer cells and promotes tumour growth. The lymphocyte infiltration and the IFN-γ status may be the key to effective anti-PD-1 or anti-PD-L1 therapy in ovarian cancer.

Differential expression and the anti-apoptotic effect of human placental neurotrophins and their receptors.

Neurotrophin (NT) is important in the survival, maintenance and differentiation of neuronal tissue, and functions in follicle maturation, tumor growth, angiogenesis and immunomodulation; however, the expression of NT and its receptors (NTR) in human placenta and their influence on fetal growth are unclear. Here we investigated the correlation of NT and NTR in human placenta with uterine environment and fetal growth. TrkB, a NTR, mRNA was expressed on decidual and villous tissue and increased with gestational age, localizing in the trophoblast layer and endothelium by immunohistochemistry. Villous TrkB mRNA was significantly increased in preeclampsia (PE) than in controls and was higher in the normotensive small for gestational age (SGA) placenta, although it was not significant. It was also significantly increased in the small twin of discordant twin pregnancies. Brain-derived neurotrophic factor (BDNF), the main ligand of TrkB, was expressed in membranous chorion and villous tissue and was significantly higher in maternal plasma in normotensive SGA and PE than in controls. TrkB mRNA expression was up-regulated on cultured villous tissue explants and on JEG-3, a choriocarcinoma cell line, by H(2)O(2) treatment. BDNF decreased apoptotic cells in H(2)O(2)-treated JEG-3, indicating that BDNF/TrkB signaling had anti-apoptotic effects against oxidative stress in JEG-3, suggesting a protective role of BDNF/TrkB in human villous tissue under unfavorable conditions in utero.

Randomized phase II study comparing docetaxel plus cisplatin, docetaxel plus carboplatin, and paclitaxel plus carboplatin in patients with advanced or recurrent endometrial carcinoma: a Japanese Gynecologic Oncology Group study (JGOG2041).

BACKGROUND: The purpose of this study is to assess the efficacy and safety of treatment with taxane plus platinum in combination therapies for advanced or recurrent endometrial carcinoma. PATIENTS AND METHODS: Patients with measurable disease derived from histologically confirmed stage III/IV or recurrent endometrial carcinoma were randomly assigned to receive docetaxel plus cisplatin (DP), docetaxel plus carboplatin (DC), or paclitaxel plus carboplatin (TC) every 3 weeks until disease progression or adverse events prohibited further therapy. Among these regimens, the study evaluated the tumor response rate as the primary end point as well as toxicity. RESULTS: Ninety patients were enrolled. Of them, 88 were eligible and consequently 29, 29, and 30 patients were randomly assigned to DP, DC, and TC, respectively. Tumor response rates were 51.7%, 48.3%, and 60.0% in DP, DC, and TC, respectively (P = 0.65). The following toxic effects were observed: grade 3/4 neutropenia in 83.3%, 90.0%, and 76.6%; febrile neutropenia in 10.0%, 6.7%, and 3.3%; grade 3/4 thrombocytopenia in 6.7%, 10.0%, and 10.0%; grade 3/4 diarrhea in 13.3%, 3.3%, and 0%, respectively; and grade 3 neurotoxicity in 10.0% of TC. These toxicity profiles were not significantly different. CONCLUSION: The taxane plus platinum combination therapies could be candidates in further phase III trials for endometrial carcinoma.

Prenatal treatment of meconium peritonitis with urinary trypsin inhibitor.

We describe a case of congenital meconium peritonitis with progressive fetal ascites and polyhydramnios. Fetal ascites could be only partially reduced on paracentesis at 29 weeks' gestation, and it subsequently increased. Urinary trypsin inhibitor (UTI), a physiological anti-inflammatory substance, was administered into the fetal abdominal cavity at a second paracentesis performed at 35 weeks' gestation. There was a significant amount of fetal ascites remaining 1 day after the second paracentesis, but this completely resolved within 5 days. A healthy infant was delivered vaginally and no surgical intervention was required. The case suggests that UTI can reduce meconium-induced chemical peritonitis and thereby facilitate intrauterine remission of fetal ascites.

Prenatal diagnosis of anterior sacral meningocele.

Anterior sacral meningocele is an extremely rare condition and there has been only one previous report of a prenatal diagnosis. We report the case of a 36-year-old primigravida who was referred following detection of a huge fetal pelvic cyst on routine ultrasound examination at 19 + 4 weeks' gestation. Neither fetal ultrasound nor magnetic resonance imaging (MRI) at 20 + 5 weeks' gestation could detect communication between the cyst and the spinal cord. Because extension of the pear-shaped cyst through the pelvic diaphragm down to the perineum was reminiscent of dilated vagina and uterine cervix, a tentative diagnosis of hydrometrocolpos secondary to imperforate hymen was considered. On follow-up MRI at 33 + 5 weeks' gestation, a narrow stalk connecting the pelvic cyst and the spinal canal through the anterior sacral foramen was clearly delineated, allowing us to reach the prenatal diagnosis of anterior sacral meningocele.

Branched-chain amino acid supplemented diet during maternal food restriction prevents developmental hypertension in adult rat offspring.

Maternal food restriction is known to cause developmental hypertension in offspring. We have previously shown that maternal high-protein diet can reverse fetal programming of hypertension and that branched-chain amino acid (BCAA) concentrations in maternal and fetal plasma were increased by maternal high-protein intake. Then, we hypothesized that isocaloric supplementation with BCAA to a maternal food restriction can reverse the adverse outcome. Pregnant rats were divided into four groups at 7.5 days postcoitum: normally nourished (NN) and 70% undernourished (UN) groups with and without BCAA supplementation (NN-standard diet (SD), NN-BCAA, UN-SD and UN-BCAA groups). Compared with pups in the NN groups, those in the UN-SD group had significantly increased systolic blood pressure (SBP) at 8 and 16 weeks of age (P < 0.05). However, the elevation of SBP was not observed in offspring in the UN-BCAA group. Offspring glomeruli number of the UN groups was significantly lower (P < 0.05) than that of the NN groups, independent of BCAA supplementation. Angiotensin II receptor type 2 (ATR2) mRNA and protein expression in the kidney was significantly augmented in the UN-BCAA group at 30 weeks of age. In conclusion, BCAA supplementation during maternal food restriction prevents developmental hypertension together with increased ATR2 expression in adult offspring kidney.

Identification of an ovarian clear cell carcinoma gene signature that reflects inherent disease biology and the carcinogenic processes.

Ovarian clear cell carcinoma (OCCC) shows unique clinical features including an association with endometriosis and poor prognosis. We previously reported that the contents of endometriotic cysts, especially high concentrations of free iron, are a possible cause of OCCC carcinogenesis through iron-induced persistent oxidative stress. In this study, we conducted gene expression microarray analysis using 38 ovarian cancer cell lines and identified genes commonly expressed in both OCCC cell lines and clinical samples, which comprise an OCCC gene signature. The OCCC signature reproducibly predicts OCCC specimens in other microarray data sets, suggesting that this gene profile reflects the inherent biological characteristics of OCCC. The OCCC signature contains known markers of OCCC, such as hepatocyte nuclear factor-1beta (HNF-1beta) and versican (VCAN), and other genes that reflect oxidative stress. Expression of OCCC signature genes was induced by treatment of immortalized ovarian surface epithelial cells with the contents of endometriotic cysts, indicating that the OCCC signature is largely dependent on the tumor microenvironment. Induction of OCCC signature genes is at least in part epigenetically regulated, as we found hypomethylation of HNF-1beta and VCAN in OCCC cell lines. This genome-wide study indicates that the tumor microenvironment induces specific gene expression profiles that contribute to the development of distinct cancer subtypes.

Pregnancy complicated with Buerger's disease.

Buerger's disease is an inflammatory occlusive vascular disorder involving small- and medium-sized arteries in the distal extremities and is usually complicated with thrombophlebitis. Since Buerger's disease develops most frequently in men who smoke, pregnancy complicated with this disease is extremely rare. Only three pregnancies have been reported previously. All cases indicate that Buerger's disease worsens during pregnancy. However, anti-coagulant therapy appeared to be effective in this case. Accordingly, careful observation is mandatory in pregnancies complicated with Buerger's disease.

Correlation between MRI and histopathologic findings in stage I cervical carcinomas: influence of stromal desmoplastic reaction.

Although the effectiveness of magnetic resonance imaging (MRI) in depicting cervical carcinoma has been reported, whether MRI can detect early-stage or stage IB "occult"-type cervical carcinoma remained undetermined. We examined the correlation between MRI and pathologic findings in 38 stage I (IB 28 cases, IA 10 cases) cervical carcinoma patients, with special reference to the influence of desmoplastic stromal reaction around the tumor. The results demonstrated that the tumor was detected by MRI in none of stage IA patients but in 21 (75%) stage IB patients. The image was clearly demonstrated in 15 of 18 (83%) tumors of more than 2 cm in diameter and in 6 of 10 (60%) tumors of 2 cm or less. The tumor image was evident in 21 of 22 (95%) tumors with prominent (>200 micron) stromal reaction but in none of 6 tumors with minimal (

Gonadotropins and ovarian carcinogenesis: a new era of basic research and its clinical implications.

The possible relationship between gonadotropins and ovarian carcinoma development has received much attention, and in recent years, great progress has been made in basic and epidemiologic research about this issue. Gonadotropins sensitivity in the ovarian surface epithelium (OSE) and in a subset of ovarian carcinomas has been established in vivo and in vitro. Gonadotropins have been shown to induce various biologic actions in the OSE and ovarian carcinoma cells, such as changes in cell proliferation, apoptosis, cell adhesion, and chemosensitivity. These basic studies strongly suggest that gonadotropins are involved in the development and progression of ovarian carcinoma. In contrast, although earlier studies showed a significant risk of infertility therapy for ovarian carcinoma development, subsequent studies reported only slightly increased or no significant increased risk of gonadotropin stimulation and/or assisted reproductive technologies for ovarian carcinoma development. Therefore, the association between ovarian stimulation and ovarian carcinoma remains controversial. Nevertheless, since development of ovarian carcinoma in infertile women during infertility treatment is a serious concern for gynecologists, this review also covers important points for clinical practice, especially the issue of early detection of ovarian carcinoma.

Vulvar Paget's disease with underlying adenocarcinoma simulating breast carcinoma: case report and review of the literature.

We report a case of extramammary Paget's disease with underlying adenocarcinoma simulating breast carcinoma of the vulva. An 82-year-old woman was found to have a 5 x 3-cm bulky tumor located in the left labium major, infiltrating to the clitoris, left labium minor, and left lateral tissue of the vulva. Small biopsy of the vulva showed intraepidermal proliferation of Paget cells. The patient underwent wide local excision of the vulvar tumor and dissection of left inguinal lymph nodes. Histopathological examination of the resected specimens revealed that Paget cells were distributed singly or tended to form small nests in the epidermis, and that association of these cells with the underlying carcinoma invading to the subcutis could be seen. The underlying carcinoma was composed of squamoid solid nests with central necrotic debris, mimicking 'comedocarcinoma' of the breast. In other areas, the tumor cells were present in tubular formations and solid cords reminiscent of invasive ductal carcinoma of the breast. Immunohistochemically, the Paget cells and the underlying carcinoma cells were positive for carcinoembryonic antigen, epithelial membrane antigen, estrogen receptors, and glandular keratins except for CK 20. We speculate that our case is vulvar Paget's disease presenting as a manifestation of underlying breast carcinoma of the vulva, which might have arisen from either the ectopic breast tissue or anogenital mammary-like glands.

Successful treatment with stereotactic radiosurgery for brain metastases of endometrial carcinoma: a case report and review of the literature.

Brain metastases from endometrial carcinomas are rare and the treatment is usually difficult. Here, we report a patient with stage IV endometrial carcinoma whose brain metastases showed complete remission after stereotactic radiosurgery using a gamma-knife. A 48-year-old woman underwent removal of a single brain metastatic lesion, and one month later underwent hysterectomy for endometrioid-type G3, endometrial adenocarcinoma. After hysterectomy, a cranial magnetic resonance imaging (MRI) demonstrated multiple brain metastases and the patient received two courses of stereotactic radiosurgery and six courses of chemotherapy. Complete response of the brain lesions was obtained, and she is well without recurrence 38 months after the second stereotactic radiosurgery.

Expression and activation of MAP kinases, ERK1/2, in the human villous trophoblasts.

Mitogen-activated protein kinase (MAP kinase) plays a central role in the signal transduction for diverse cellular responses, such as proliferation, differentiation, stress response and cell death, via activation after binding of growth factors to the respective receptors on the cell membrane. In the human placental tissues, however, little is known about the expression and activation of the classical MAP kinases, extracellular signal-regulated kinase1/2 (ERK1/2). We therefore examined the expression of ERK1/2 in the human chorionic and placental tissues between 5 and 41 weeks of gestation, using Western blotting, immunohistochemistry and in situ hybridization. To explore the activation of ERK1/2 protein, we used an antibody that reacts with both phosphorylated and non-phosphorylated ERK1/2 (total ERK1/2), as well as antibodies that react only with phosphorylated ERK1/2. The expression pattern of phosphorylated ERK1/2 in the trophoblasts was compared with that of various growth factor receptors, such as c-met, IGF-1R, flt-1, EGFR, PDGFR, Bek, and flg. Total ERK1/2 was immunolocalized in the villous cytotrophoblasts (CTs), but not in the syncytiotrophoblasts (STs), throughout pregnancy. In situ hybridization also showed the localization of ERK1 mRNA in the villous CTs. Interestingly, however, phosphorylated ERK1/2 was immunolocalized in the villous CTs only up to 12 weeks of gestation. Western blot also showed the stronger bands of phosphorylated ERK1/2 in the tissues of the first trimester. Among the growth factor receptors, c-met was strongly expressed in the villous CTs during the first trimester, and resembled the expression pattern of phosphorylated ERK1/2. These findings suggest that the MAP kinase pathway is activated in the villous CTs during the first trimester in the human placenta.

Expression of the cell cycle regulator p27(Kip1) in normal squamous epithelium, cervical intraepithelial neoplasia, and invasive squamous cell carcinoma of the uterine cervix. Immunohistochemistry and functional aspects of p27(Kip1).

BACKGROUND: Abnormality of cell cycle regulators and tumor suppressors, such as cyclin dependent kinase inhibitors (cdkIs), has been reported in malignant tumors. The current study was undertaken to examine the involvement of a cdkI, p27(Kip1) (p27), in the neoplastic process of the uterine cervical epithelium. METHODS: Immunohistochemical staining of p27 was performed in samples of normal cervical tissue (30 samples), cervical intraepithelial neoplasias (CINs; 17 samples), and invasive squamous cell carcinoma (SCC; 25 samples). The results were compared with the expression levels of Ki-67, cdk2, and cyclin E. The functional aspects of the p27 protein, such as its ability to bind to cdk2 and the phosphorylation activity of p27-bound cdk2, also were evaluated with an immunoprecipitation and histone H1 kinase assay. RESULTS: In normal cervical epithelia, the expression of p27 was strong in the intermediate and superficial cells but very weak in the parabasal cells. In CIN samples, the expression of p27 was negligible. The expression of p27 in these tissues showed an inverse topologic correlation to that of Ki-67, cdk2, and cyclin E. However, it is noteworthy that the number of p27 positive cells increased in SCC samples that also showed increased expression of Ki-67, cdk2, and cyclin E. The p27 protein in SCC samples was bound to cdk2 and cyclin E. However, cdk2 that was bound to p27 still possessed histone H1 kinase activity. CONCLUSIONS: The expression of p27 may be involved in the growth regulation of the normal squamous epithelium in the uterine cervix. However, aberrant function of p27 expression may occur in invasive SCC of the cervix.

Cortical mapping of gait in humans: a near-infrared spectroscopic topography study.

While we have a fair understanding of how and where forelimb-hand manipulative movements are controlled by the neocortex, due to functional imaging studies, we know little about the control of bipedal movements such as walking because of technical difficulties. We succeeded in visualizing cortical activation patterns of human gait by measuring relative changes in local hemoglobin oxygenation using a recently developed near-infrared spectroscopic (NIRS) topography technique. Walking activities were bilaterally associated with increased levels of oxygenated and total hemoglobin in the medial primary sensorimotor cortices and the supplementary motor areas. Alternating foot movements activated similar but less broad regions. Gait imagery increased activities caudally located in the supplementary motor areas. These findings provide new insight into cortical control of human locomotion. NIRS topography might be also useful for evaluating cerebral activation patterns during pathological gait and rehabilitative intervention.