Origin, phenotype and autoimmune potential of T cells in human immune system mice receiving neonatal human thymus tissue.

Robust human immune system (HIS) mice are created using human fetal thymus tissue and hematopoietic stem cells (HSCs). A HIS mouse model using neonatal human thymus tissue and umbilical cord blood (CB) HSCs (NeoHu) was recently described. We improved the model by removing the native murine thymus, which can also generate human T cells, and demonstrated definitively the capacity of human T cells to develop in a grafted neonatal human thymus. Human T cells derived from the neonatal thymus tissue appeared in peripheral blood early post-transplantation and CB-derived T cells appeared later. Naïve T cells were demonstrated in peripheral blood but effector memory and T peripheral helper phenotypes predominated later, in association with development of autoimmunity in some animals. Treatment of thymus grafts with 2-deoxyglucose (2-DG) increased the proportion of stem cells derived from injected HSCs, delayed onset of autoimmune disease, reduced early T cell reconstitution, and reduced effector/memory T cell conversion. Younger neonatal human thymus tissue was associated with improved T cell reconstitution. While the NeoHu model bypasses the need for fetal tissue, it has yet to demonstrate equivalent reconstitution to fetal tissue, though 2-DG can improve results by removing native thymocytes prior to transplantation.

Biodistribution, pharmacokinetics and radioimmunotherapy of 188Re-cetuximab in NCI-H292 human lung tumor-bearing nude mice.

Background Cetuximab is a fully humanized IgG1 subclass monoclonal that binds specifically to the human epidermal growth factor receptor (EGFR). Although EGFR is expressed in normal cells, the overexpression of EGFR is detected in many human cancers, such as colon, rectum and lung tumors. In this study, cetuximab with a combination of radiotherapy nuclear 188Re achieved better therapeutic effect on lung cancer. Methods188Re-cetuximab administered by the i.v. route in human NCI-H292 lung tumor-bearing mice was investigated. NanoSPECT/CT images were taken to evaluate the distribution and tumor targeting of 188Re-cetuximab in mice. The anti-tumor effect of 188Re-cetuximab was assessed by the tumor growth inhibition, survival ratio. Results For nanoSPECT/CT imaging, a significant uptake in tumor was observed at 24 and 48 h following the injection of 188Re-cetuximab. The anti-tumor effect of 188Re-cetuximab was assessed by tumor growth inhibition and the survival ratio. The tumor-bearing mice treated with 188Re-cetuximab showed a better mean tumor growth inhibition rate (MGI = 0.049) and longer median survival time and lifespan (62.50 d; 70.07%) than those treated with 188Re-perrhenate and cetuximab only by single injection. A synergistic effect of tumor growth inhibition was observed with the combination index exceeding one for 188Re-cetuximab (CI = 6.135 and 9.276). Conclusion The tumor targeting and localization of 188Re-cetuximab were confirmed in this study. Synergistic therapeutic efficacy was demonstrated for the radioimmunotherapy of 188Re-cetuximab. The results of this study reveal the potential advantage and benefit obtained from 188Re-cetuximab for diagnosis and therapy of oncology applications in the future.

Radiolabeling, Characteristics and NanoSPECT/CT Imaging of 188Re-cetuximab in NCI-H292 Human Lung Cancer Xenografts.

BACKGROUND/AIM: Cetuximab has exhibited high EGFR-targeting specificity and clinical promise in previous studies. In this study, we formulated unit dose kits for preparation of high specific activity 188Re-cetuximab for imaging and treatment of EGFR-positive cancer. MATERIALS AND METHODS: 188Re-cetuximab was prepared by adding 0.37-0.74 GBq/0.5 ml of 188Re-perrhenate for 4 h at 37°C. Cell surface expression of EGFR, cell binding and cytotoxic effects were evaluated in vitro using both EGFR-positive (NCI-H292, A431) and EGFR-negative (BT483) tumors. A nanoSPECT/CT imaging study was performed in mice bearing EGFR-expressing NCI-H292 tumors. RESULTS: 188Re-cetuximab bound specifically to EGFR-expressing cells and labeling of radionuclides to cetuximab preserved the binding ability of the antibody. Besides, the cytotoxic effect of 188Re-cetuximab was increased dose-dependently. NanoSPECT/CT imaging revealed that 188Re-cetuximab could continually target the tumor region for at least 48 h. CONCLUSION: The highly specific targeted property of 188Re-cetuximab suggested that it is suitable as a diagnostic tool and maybe a potent radioimmunotherapy agent in EGFR-positive cancers.

Numerical modeling of nanodrug distribution in tumors with heterogeneous vasculature.

The distribution and accumulation of nanoparticle dosage in a tumor are important in evaluating the effectiveness of cancer treatment. The cell survival rate can quantify the therapeutic effect, and the survival rates after multiple treatments are helpful to evaluate the efficacy of a chemotherapy plan. We developed a mathematical tumor model based on the governing equations describing the fluid flow and particle transport to investigate the drug transportation in a tumor and computed the resulting cumulative concentrations. The cell survival rate was calculated based on the cumulative concentration. The model was applied to a subcutaneous tumor with heterogeneous vascular distributions. Various sized dextrans and doxorubicin were respectively chosen as the nanodrug carrier and the traditional chemotherapeutic agent for comparison. The results showed that: 1) the largest nanoparticle drug in the current simulations yielded the highest cumulative concentration in the well vascular region, but second lowest in the surrounding normal tissues, which implies it has the best therapeutic effect to tumor and at the same time little harmful to normal tissue; 2) on the contrary, molecular chemotherapeutic agent produced the second lowest cumulative concentration in the well vascular tumor region, but highest in the surrounding normal tissue; 3) all drugs have very small cumulative concentrations in the tumor necrotic region, where drug transport is solely through diffusion. This might mean that it is hard to kill tumor stem cells hiding in it. The current model indicated that the effectiveness of the anti-tumor drug delivery was determined by the interplay of the vascular density and nanoparticle size, which governs the drug transport properties. The use of nanoparticles as anti-tumor drug carriers is generally a better choice than molecular chemotherapeutic agent because of its high treatment efficiency on tumor cells and less damage to normal tissues.

Postpartum Acute Kidney Injury: Experience of a Tertiary Care Center.

Pregnancy-related-acute kidney injury (PR-AKI) had decreased from 40% to 20% in 1960 to <10% in recent series, mostly due to meticulous antenatal management. Postpartum-AKI (PP-AKI) resulting from late obstetric complications has become more apparent after improvement in antenatal care and legalization of medical termination of pregnancy. Women with renal injury in peripartum period admitted to our hospital over a period of 2 years (April 2013 to May 2015) were studied. Of 713 patients of AKI admitted, 61 had PR-AKI with an incidence of 4.27%. Out of the 61 patients, 28 had PP-AKI with an incidence of 1.96%. The mean age of patients with PP-AKI was 26.10 ± 4.3 years. Sepsis was the most common cause accounting for 11 (39.28%) cases followed by postpartum hemorrhage (PPH) in 7 (25%) cases. Renal biopsy was done in nine patients, out of whom four were having cortical necrosis. Patients having diffuse cortical necrosis remained dialysis-dependent. High contribution of sepsis and PPH to PP-AKI in our setting makes it an ideal target for rectification. Protocolized peripartum monitoring and standard clinical practices of asepsis will go long way in decreasing the incidence of PP-AKI and maternal morbidity in our valley.

Erratum to: 2016 WSES guidelines on acute calculous cholecystitis.

[This corrects the article DOI: 10.1186/s13017-016-0082-5.].

2016 WSES guidelines on acute calculous cholecystitis.

Acute calculus cholecystitis is a very common disease with several area of uncertainty. The World Society of Emergency Surgery developed extensive guidelines in order to cover grey areas. The diagnostic criteria, the antimicrobial therapy, the evaluation of associated common bile duct stones, the identification of "high risk" patients, the surgical timing, the type of surgery, and the alternatives to surgery are discussed. Moreover the algorithm is proposed: as soon as diagnosis is made and after the evaluation of choledocholitiasis risk, laparoscopic cholecystectomy should be offered to all patients exception of those with high risk of morbidity or mortality. These Guidelines must be considered as an adjunctive tool for decision but they are not substitute of the clinical judgement for the individual patient.

Relationship between Serum Soluble Interleukin-2 Receptor and Renal Allograft Rejection: A Hospital-Based Study in KashmirValley.

BACKGROUND: Even after adequate immunosuppression therapy, acute rejection continues to be the single most important cause of graft dysfunction after renal transplantation. Renal allograft biopsy continues to be the reference standard, though certain clinical and biochemical parameters are helpful in assessment of these patients. Renal allograft rejection is mediated by T lymphocytes, expressing cell surface interleukin-2 receptors (IL-2R) which has been suggested as a marker of acute rejection episodes after organ transplantation. OBJECTIVE: To determine the pre- and post-transplantation serum soluble IL-2R levels in live related kidney transplant patients to predict acute rejection episodes. METHODS: Serial serum samples from 75 recipients and 41 healthy controls were assessed for soluble IL-2R levels by ELISA. The outcome of the graft was also determined for each recipient. RESULTS: The mean±SD serum soluble IL-2R levels in renal allograft recipients with rejection were significantly (p<0.001) higher than those without rejection (329.85±59.22 vs 18.12±11.22 pg/mL). The elevation of serum soluble IL-2R was evident in acute rejection episodes and found before elevation of serum creatinine. The higher values of serum soluble IL-2R in the rejection group were significantly reduced after recovery of allograft function by adequate anti-rejection therapy. 36.4% of patients in the rejection group had proven positive biopsies for the rejection and higher creatinine values, which was found to be statistically significant (p<0.001). A cohort of 41 healthy controls showed significantly (p<0.05) lower serum soluble IL-2R concentrations (15.27±7.79 pg/mL) when compared with the rejection group. CONCLUSION: Serum soluble IL-2R concentrations showed significant correlation with the acute rejection episodes in the renal allograft recipients. Prediction of soluble IL-2R levels might help the early detection of rejection episodes, which may pave way for the management of immunosuppression regimes and better graft functioning.

Single nucleotide polymorphisms of the maternal Msx2 gene and their association with fetal neural tube defects in Han ethnic group in Shanxi Province, China.

BACKGROUND: Neural tube defects are the most common human birth defects. The causes are multifactorial with complex genetic and environmental factors, although the exact genetic causes are unknown. This research was conducted to study the frequency of Msx2 gene polymorphisms in 59 women with a history of pregnancy with a neural tube defect and in 73 healthy controls. We aimed to determine the effect of this genetic polymorphism on the incidence of neural tube defects in the Han Chinese population. METHODS: We studied 59 mothers with at least one previous child with a neural tube defect (the case group) and 73 case-control subjects during the same period, from Shanxi Province, China. We analyzed the genotypic distributions and allele frequencies of Msx2 C386T polymorphisms in DNA samples from the case and control groups. A three-dimensional protein model was predicted using Swiss-Pdb Viewer software version 4.0. Disease association was analyzed using chi-square tests. RESULTS: Significant differences were observed in the genotypes and allele frequencies of the Msx2 C386T allele between the case and control groups (CT: 32% vs. 15%, P = 0.0073 and TT 15% vs. 4%, P = 0.013, respectively). Logistic regression analysis showed that the C386T mutation is a potential risk factor for neural tube defects (P < 0.05; OR: 3.466; 95%CI: 1.831 - 6.560). Three-dimensional structure prediction revealed that the Msx2 C386T mutation results in a threonine substitution for methionine at position 129 of exon 2, which might lead to structural mutations or dysfunctions in the protein encoded by Msx2. CONCLUSION: Maternal Msx2 C386T gene polymorphisms were associated with fetal neural tube defects in Han Chinese women in Shanxi Province.

Peritoneal hydatidosis in a young girl.

We report a case of peritoneal hydatidosis that occurred post laparotomy. Patient was diagnosed nine months after she had laparotomy for suspected acute appendicitis. The whole peritoneal cavity was studded with cysts. In view of diffuse involvement, patient was managed conservatively and showed response to medical therapy.

Hepatocyte gene transfer mediated by stable polyplexes based on MPP-containing DNA complexes.

BACKGROUND: In the field of gene therapy, viral vectors as delivery tools have a number of disadvantages for medical application. This study aimed to explore a novel nonviral vector as a vehicle for gene therapy. METHODS: Transvector-rpE-MPP and EGFP (enhanced green fluorescent protein) were used as the gene transfer carrier and the reporter gene, respectively. Polyplexes which integrate transvector-rpE-MPP, the object gene, and EGFP were formed. The optimal charge ratio, stability, and transduction capacity of the polyplexes in mouse hepatocytes in vitro and in mouse liver in vivo were investigated. The polyplexes of transvector-rpE-MPP and pcDNA(3)-EGFP, with charge ratios of 0, 0.25, 0.5, 0.75, 1 and 1.5 were compared to determine the optimal charge ratio. RESULTS: Polyplexes with charge ratios of 1:1 were most stable; pcDNA(3)-EGFP in these complexes resisted digestion by DNase I and blood plasma. On the other hand, pcDNA(3)-EGFP alone was digested. Fluorescence analysis indicated that transvector-rpE-MPP successfully delivered the reporter gene EGFP into hepatocytes and that EGFP expression was detected in hepatocyte cultures and in liver tissue. CONCLUSION: These results have laid a foundation for further study of a novel nonviral gene delivery system.

Multiple myeloma--presenting as acute kidney injury.

BACKGROUND: Multiple myeloma (MM) is a commonly encountered hematological malignancy with significant renal involvement and often presents as renal failure. The aim of the present study is to analyze clinical spectrum of acute renal failure (ARF) in patients with MM. MATERIAL AND METHODS: We analyzed 26 (males 24; females 2) patients of multiple myeloma who were referred for evaluation of ARF between July 1994 - June 2007. The referral diagnosis did not include MM in majority 23 (88%) of the patients. Multiple myeloma was diagnosed by at least two of the four features; (1) lytic bone lesions, (2) serum or urine monoclonal peak, (3) Bence Jones proteinuria and (4) more than 20% plama cells in marrow aspirate. RESULTS: Multiple myeloma contributes 1.93% of total ARF cases (26/1342) over a period of thirteen years. Mean age of patients was 59.3 +/- 7.4 years. The clinical manifestations of myeloma included; anemia (100%), Bence Jones proteinuria (80%), "M" peak in serum electrophoresis (69%), lytic bone lesions (62%), "M" peak in urine electrophoresis (54%), body pain (58%), plasma cells more than 20% in bone marrow aspirate (38%). Oliguric ARF was seen in 73% patients. The precipitating factors of ARF identified were; hypercalcemia (31%); infection (23%); volume depletion (19%); and NSAIDs in (15%). Dialysis support was needed in 77% of the patients because of severe renal failure at presentation with mean serum creatinine of 9.05 +/- 2.84 mg%. Seventeen patients completed chemotherapy, seven last to follow up and two patients died. Ten (38.5%) patients had complete recovery of renal function; three patients had partial recovery and off dialysis and four patients remained dialysis dependent. Remission of myeloma was achieved in nine of seventeen patients treated with chemotherapy Renal biopsy finding in nine patients revealed-cast nephropathy in (4), amyloidosis in (3), proliferative glomerulonephritis in (1) and cast nephropathy with chronic interstitial nephritis and plasma cell infiltration in one patient. CONCLUSION: Acute reversible renal failure is a common complication in MM, multiple myeloma should be considered as cause a cause of unexplained ARF in middle aged and elderly patients.

Calmodulin kinase II activation of mitogen-activated protein kinase in PC12 cell following all-trans retinoic acid treatment.

Previous studies have shown that apoptosis can be mediated by activation of either calmodulin kinase II (CaMKII) or mitogen-activated protein kinase (MAPK), ERK and p38. In the present study, we investigated whether CaMKII is involved in activation of ERK and p38 in response to all-trans retinoic acid (ATRA) treatment in PC12 cells. Results showed that ATRA-induced activation of ERK and p38 occurred later than that of CAMKII. Knockdown of CAMKII by siRNA significantly suppressed ATRA-induced activation of ERK and p38. These results demonstrated that activation of ERK and p38 following ATRA exposure is CAMKII-dependent. Treatment with ATRA also resulted in cell death characterized by apoptosis in PC12 cells. Results suggest that CaMKII-dependent activation of ERK and p38 is related to apoptotic cell death.

p38 mitogen-activated protein kinase-dependent regulation of SRC-3 and involvement in retinoic acid receptor alpha signaling in embryonic cortical neurons.

Appropriate retinoic acid (RA) signaling is essential in the development of the central nervous system (CNS). Previous studies have shown that RA activates p38 mitogen-activated protein kinase (MAPK) and steroid receptor coactivator (SRC)-3 in tumor cells in vitro. It is unknown whether the activation of p38 MAPK and SRC-3 is involved in RA-mediated CNS development. The current study investigated a possible role for p38 MAPK in the regulation of (SRC)-3 phosphorylation/degradation and in retinoic acid receptor (RAR)alpha signaling in mouse fetal cortical neurons treated with all-trans retinoic acid (ATRA). Results showed that ATRA treatment rapidly activated p38 MAPK, which in turn resulted in phosphorylation with subsequent degradation of SRC-3. Inhibition of p38 MAPK by SB203580 blocked the phosphorylation and degradation of SRC-3. The binding of RARalpha to retinoic acid responsive element (RARE) was rapidly increased in neurons following ATRA treatment. Inhibition of p38 MAPK significantly enhanced the RARalpha-RARE binding activity, but had no effects on ATRA-induced decrease of RARalpha. Treatment of the fetal cortical neurons with ATRA significantly increased the expression of HOXd3, a retinoid-target gene. The increase of HOXd3 expression was augmented when p38 MAPK activity was inhibited by a specific inhibitor, SB203580. Results suggest that ATRA activates the p38 MAPK signal pathway with resultant degradation of SRC-3, and that p38 MAPK is involved in the regulation of RARalpha-mediated signaling in developing neurons.

Renal disease is a prodrome of multiple myeloma: an analysis of 50 patients from eastern India.

This study describes a spectrum of renal diseases that can precede the diagnosis of multiple myeloma (MM). Patients presenting manifestations of renal disease were recorded as individual patients of MM. Fifty patients (male 41; female 9) were included in this study. Diagnosis of MM was confirmed by two or more of the following four features: lytic bone lesions, serum or urine monoclonal peak, Bence Jones proteinuria, and greater than 20% plasma cells in bone marrow. Renal disease was present in 42 of 50 (84%) patients before MM was diagnosed. In only eight of 50 (16%) patients, diagnosis of MM preceded the detection of renal disease. Renal diseases consisted of acute renal failure in 26 patients (52%), chronic renal failure in 15 patients (30%) and nephrotic syndrome in 9 patients (18%). Some of the patients with acute or chronic renal failure also had heavy proteinuria. Percutaneous renal biopsy was done in 17 patients. Renal histopathology showed amyloidosis (n = 10), cast nephropathy (n = 5), nodular glomerulosclerosis (n = 1), and mesangioproliferative glomerulonephritis with plasma cell infiltration (n = 1). Hypercalcemia (calcium 11-13.8 mg/dL) was the most common precipitating factor for acute renal failure. All 50 patients received combination chemotherapy of melphalan and prednisolone or vincristine, Adriamycin, and dexamethasone. More than half of the total number of patients did not complete chemotherapy because of death or lost to follow-up. Nineteen patients with acute renal failure and eight patients with chronic renal failure were treated with hemodialysis. Fourteen patients (28%) with acute renal failure had recovery of renal function. Twenty-three patients (46%) were lost to follow-up. Seven patients (14%) died from sepsis, uremia, or hyperkalemia. Remission of MM was found in 9 of 21 (42.8%) patients who completed chemotherapy. Thus, acute renal failure is the most common renal disease preceding the diagnosis of MM. Reversal of renal function is achieved with chemotherapy and hemodialysis treatment.

What grabs his attention but not hers? Estrogen correlates with neurophysiological measures of vocal change detection.

Prior research revealed sex differences in the processing of unattended changes in speaker prosody. The present study aimed at investigating the role of estrogen in mediating these effects. To this end, the electroencephalogram (EEG) was recorded while participants watched a silent movie with subtitles and passively listened to a syllable sequence that contained occasional changes in speaker prosody. In one block, these changes were neutral, whereas in another block they were emotional. Estrogen values were obtained for each participant and correlated with the mismatch negativity (MMN) amplitude elicited in the EEG. As predicted, female listeners had higher estrogen values than male listeners and showed reduced MMN amplitudes to neutral as compared to emotional change in speaker prosody. Moreover, in both, male and female listeners, MMN amplitudes were negatively correlated with estrogen when the change in speaker prosody was neutral, but not when it was emotional. This suggests that estrogen is associated with reduced distractibility by neutral, but not emotional, events. Emotional events are spared from this reduction in distractibility and more likely to penetrate voluntary attention directed elsewhere. Taken together, the present findings provide evidence for a role of estrogen in human cognition and emotion.

Correlation of polymorphism of MTHFRs and RFC-1 genes with neural tube defects in China.

BACKGROUND: Maternal periconceptional supplementation of folate reduces the incidence of neonatal Neural Tube Defects, indicating that changes in folate metabolism play a role in formation of NTDs. The mutations on two genes involved in folate metabolism, the C677 of the MTHFR gene and the RFC-1(A80G) gene are potential risk factors of NTDs. METHODS: In this study, we analyzed the genotypic distributions and allele frequencies of MTHFR C677T and RFC-1 A80G polymorphisms in DNA samples from mothers with at least one previous child with NTDs (the NTD group) and controls. RESULTS: Our results indicated that there was a significant difference in the genotype and allele frequencies of RFC-1 80A-->G between the NTD group and controls (p = .008 and p = .017, respectively). There was, however, no significant difference in the genotype and allele frequencies of the MTHFR 677C-->T polymorphism between the NTD group and controls. The NTD group was further separated into the upper and lower types by location of abnormalities. The frequency of RFC-1 80A/G and 80G/G was significantly higher in the upper group than the control (p = .009 and p = .005, respectively). The frequency of G-alleles was also significantly higher in the upper group than the control (OR 2.42; p = .006; 95% CI: 1.28-4.58). For the MTHFR C677 gene, the frequency of T-alleles was significantly lower in the lower defect type than the control group (OR 0.32; p = .027; 95% CI: 0.11-0.9). CONCLUSIONS: These results suggest that in the Shanxi population RFC-1 polymorphisms may play a role in NTD risk, whereas the impact of MTHFR C677T polymorphisms requires further clarification. Birth Defects Research (Part A) 2008.

Decreasing incidence of renal cortical necrosis in patients with acute renal failure in developing countries: a single-centre experience of 22 years from Eastern India.

BACKGROUND: Renal cortical necrosis (RCN) accounts for 2% of all cases of acute renal failure (ARF) in adults and 15-20% of ARF during the third trimester of pregnancy in developed nations. However, RCN incidence is higher in developing countries ranging from 6-7% of all cases of acute renal failure. The present study describes changing trends in the clinical spectrum of RCN in patients with ARF in Eastern India. METHODS: Patients with ARF suspected to have RCN on clinical grounds underwent percutaneous renal biopsy. Patients showing cortical necrosis on histology were included in the present study. Diffuse and patchy cortical necrosis was classified based on standard histological criteria. The patients with cortical necrosis were studied over a period of 22 years; from July 1984 to December 2005. The results of our observation were compared with respect to etiology, incidence, prognosis and outcome of renal cortical necrosis in two study periods; namely, 1984-1994 and 1995-2005. RESULTS: The incidence of RCN was 3.12% of all cases of ARF of diverse etiology. RCN was observed in 57 patients; obstetric 32 (56.2%); non-obstetric 25 (43.8%). Diffuse cortical necrosis was the dominant lesion in 41 (71.9%) patients and the remaining 16 (28%) patients had patchy cortical necrosis. The overall incidence of RCN in obstetric ARF was 15.2%; the incidence being higher (11.9%) in the post-abortal group in comparison to 3.3% in late pregnancy. RCN had occurred complicating abruptio placentae, puerperal sepsis and postpartum haemorrhage (PPH) in late pregnancy, while septic abortion was the sole cause of RCN in early pregnancy. Haemolytic uraemic syndrome (HUS) was the major (31.5%) cause of RCN in the non-obstetric group and miscellaneous factors were responsible in seven (12.3%) patients. Partial recovery of renal function was observed in 11 (19.2%), and 16 (28%) patients had progressed to ESRD. The incidence of RCN decreased from 6.7% in 1984-1994 to 1.6% in 1995-2005 of total ARF cases. RCN following obstetrical complication decreased significantly; 4.7% in the 1990s to 0.5% of the total ARF cases, in the 2000s. The mortality decreased to 19% in 1995-2005 from the initial high mortality of 72% in 1984-1994. The renal prognosis improved as a result of the decreased mortality of patients. CONCLUSION: We observed a decreasing trend in the incidence of RCN in patients with ARF in recent years, which is associated with increased patient survival and better renal prognosis. This improvement was mainly due to declining incidence and severity of RCN in obstetrical ARF.