Mapping of a major genetic modifier of embryonic lethality in TGF beta 1 knockout mice.

The transforming growth factor beta 1 (TGF beta 1) signalling pathway is important in embryogenesis and has been implicated in hereditary haemorrhagic telangiectasia (HHT), atherosclerosis, tumorigenesis and immunomodulation. Therefore, identification of factors which modulate TGF beta 1 bioactivity in vivo is important. On a mixed genetic background, approximately 50% Tgfb1-/- conceptuses die midgestation from defective yolk sac vasculogenesis. The other half are developmentally normal but die three weeks postpartum. Intriguingly, the vascular defects of Tgfb1-/- mice share histological similarities to lesions seen in HHT patients. It has been suggested that dichotomy in Tgfb1-/- lethal phenotypes is due to maternal TGF beta 1 rescue of some, but not all, Tgfb1-/- embryos12. Here we show that the Tgfb1-/- phenotype depends on the genetic background of the conceptus. In NIH/Ola, C57BL/6J/Ola and F1 conceptuses, Tgfb1-/- lethality can be categorized into three developmental classes. A major codominant modifier gene of embryo lethality was mapped to proximal mouse chromosome 5, using a genome scan for non-mendelian distribution of alleles in Tgfb1-/- neonatal animals which survive prenatal lethality. This gene accounts for around three quarters of the genetic effect between mouse strains and can, in part, explain the distribution of the three lethal phenotypes. This approach, using neonatal DNA samples, is generally applicable to identification of loci that influence the effect of early embryonic lethal mutations, thus furthering knowledge of genetic interactions that occur during early mammalian development in vivo.

Production and persistence of specific antibodies in COVID-19 patients with hematologic malignancies: role of rituximab.

The ability of patients with hematologic malignancies (HM) to develop an effective humoral immune response after COVID-19 is unknown. A prospective study was performed to monitor the immune response to SARS-CoV-2 of patients with follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), chronic lymphoproliferative disorders (CLD), multiple myeloma (MM), or myelodysplastic/myeloproliferative syndromes (MDS/MPN). Antibody (Ab) levels to the SARS-CoV-2 nucleocapsid (N) and spike (S) protein were measured at +1, +3, +6 months after nasal swabs became PCR-negative. Forty-five patients (9 FL, 8 DLBCL, 8 CLD, 10 MM, 10 MDS/MPS) and 18 controls were studied. Mean anti-N and anti-S-Ab levels were similar between HM patients and controls, and shared the same behavior, with anti-N Ab levels declining at +6 months and anti-S-Ab remaining stable. Seroconversion rates were lower in HM patients than in controls. In lymphoma patients mean Ab levels and seroconversion rates were lower than in other HM patients, primarily because all nine patients who had received rituximab within 6 months before COVID-19 failed to produce anti-N and anti-S-Ab. Only one patient requiring hematological treatment after COVID-19 lost seropositivity after 6 months. No reinfections were observed. These results may inform vaccination policies and clinical management of HM patients.

Cytokine gene-modulated dendritic cells protect against allergic airway inflammation by inducing IL-10(+)IFN-gamma(+)CD4(+) T cells.

Asthma is characterized by allergen-induced airway inflammation orchestrated by Th2 cells. Dendritic cells (DCs) were found to efficiently prime naive T-helper cells. Thus, modification of DC function may be used as an ideal tool to treat allergic asthma by changing CD4(+) T-cell differentiation or suppressing Th2 development. In this study, we examined whether a DC-based vaccine can be applied to DCs modified with interleukin (IL)-10- and IL-12-expressing adenoviruses to prevent ovalbumin (OVA)-induced asthma in mice. Herein, we show that these modified DCs efficiently moderated the characteristics of asthma, including expressions of OVA-specific antibodies, airway hyperresponsiveness, eosinophilic airway inflammation, and Th2 cytokines production. Additionally, IL-10 and IL-12 gene-modified DCs enhanced the development of both T-helper type 1 (Th1) and IL-10(+)IFN-gamma(+) (interferon-gamma) double-positive T cells in vivo. In vitro-generated OVA-specific IL-10(+)IFN-gamma(+)CD4(+) T cells inhibited the proliferation of naive CD4(+) T cells, and this suppressive effect was a cell contact-dependent mechanism. Furthermore, we showed that combined cytokine-modulated DCs could alleviate established allergic airway inflammation. Taken together, these results suggest that IL-10 and IL-12 gene-modulated DCs are effective in suppressing asthmatic airway inflammation through both immune deviation and immune suppression and are a potential therapeutic approach for asthma.

Improving neural network prediction of effluent from biological wastewater treatment plant of industrial park using fuzzy learning approach.

Three types of adaptive network-based fuzzy inference system (ANFIS) in which the online monitoring parameters served as the input variable were employed to predict suspended solids (SS(eff)), chemical oxygen demand (COD(eff)), and pH(eff) in the effluent from a biological wastewater treatment plant in industrial park. Artificial neural network (ANN) was also used for comparison. The results indicated that ANFIS statistically outperforms ANN in terms of effluent prediction. When predicting, the minimum mean absolute percentage errors of 2.90, 2.54 and 0.36% for SS(eff), COD(eff) and pH(eff) could be achieved using ANFIS. The maximum values of correlation coefficient for SS(eff), COD(eff), and pH(eff) were 0.97, 0.95, and 0.98, respectively. The minimum mean square errors of 0.21, 1.41 and 0.00, and the minimum root mean square errors of 0.46, 1.19 and 0.04 for SS(eff), COD(eff), and pH(eff) could also be achieved.

Distribution of galanin immunoreactivity in the central nervous system and the responses of galanin-containing neuronal pathways to injury.

Radioimmunoassay and immunocytochemistry were used to study the distribution of galanin, a novel 29 amino acid porcine intestinal peptide, in the central nervous system of the rat and pig. The pattern of distribution was similar in the two species, with the highest concentrations of galanin-like immunoreactivity found in the neurohypophysis, hypothalamus and sacral spinal cord. Immunocytochemical studies of these regions localized galanin-like immunoreactivity to cell bodies in the paraventricular and supraoptic nuclei of the hypothalamus, to fibres in the pars nervosa and to numerous cell bodies and fibres in the dorsal horn of the spinal cord. On both gel and high pressure liquid chromatography, galanin-like immunoreactivity in rat and pig nervous tissue eluted as a single peak in a position similar to purified procine intestinal galanin standard. Surgical and pharmacological manipulations in the rat suggest the presence of galanin in afferent fibres. An increase of galanin-like immunoreactivity was observed in the sacral spinal cord of the rat following thoracic spinal cord transection. Thus galanin-like immunoreactivity in the brain is mainly localized in the hypothalamopituitary region. The decrease of galanin-like immunoreactivity in the dorsal horn of the spinal cord, following dorsal rhizotomy and pre-treatment of rats with capsaicin, indicates that many of the fibres, which are of small diameter, may well be derived from spinal sensory neurones.

Calcitonin gene-related peptide immunoreactivity in afferent neurons supplying the urinary tract: combined retrograde tracing and immunohistochemistry.

The innervation of rat and guinea pig urinary tract was examined using immunohistochemistry, radioimmunoassay and True Blue retrograde tracing techniques and was further assessed following both surgical and chemical denervation experiments. Substantial amounts of calcitonin gene-related peptide-like immunoreactivity (range 20-150 pmol/g) were detected in tissue extracts and localised to nerve fibres distributed throughout the urinary tract of both species, these being concentrated in the ureter and base of the bladder. In the guinea pig, the number and distribution pattern of calcitonin gene-related peptide-like immunoreactive nerves appeared to be identical to that of substance P-containing nerves, whereas in the rat the former predominated. Seven days after injection of the fluorescent dye True Blue into tissues of the urinary tract, retrogradely labelled cells were found in the dorsal root ganglia. These cells had a segmental distribution pattern which was specific for each of the injection sites. Thus, after injection of True Blue into the left kidney hilum a single group of labelled cells were found in the ipsilateral T10-L2 dorsal root ganglia. In contrast, injection into the left ureter produced labelled cells in two separate groups of ipsilateral ganglia (T11-L3 and L6-S1). Injection into the wall of the bladder and upper urethra resulted in bilateral labelling, with most labelled cells occurring in L6 and S1 ganglia. Approximately 90% of labelled cells in T10-L3 dorsal root ganglia displayed calcitonin gene-related peptide-like immunoreactivity, but only 60% of retrogradely labelled bladder neurons in L6-S1 ganglia were immunoreactive for this peptide. Adult guinea pigs and neonatal rats injected systemically with capsaicin subsequently exhibited a marked reduction both in the amount of calcitonin gene-related peptide immunostaining and the concentration of immunoreactive material in the urinary tract, dorsal root ganglia and spinal cord. In rats treated neonatally with capsaicin, there was a significant reduction in the number of retrogradely labelled cells and a hypertrophy of the bladder. Sectioning of the pelvic and hypogastric nerves in the rat also resulted in a depletion of calcitonin gene-related peptide-like immunoreactive nerves in the bladder, whereas chemical sympathectomy appeared to have no effect. The results indicate that calcitonin gene-related peptide immunoreactivity occurs in a major proportion of afferent neurons supplying the urinary tract of the rat and guinea pig.(ABSTRACT TRUNCATED AT 400 WORDS)

Quantitative analysis of calcitonin gene-related peptide- and neuropeptide Y-immunoreactive nerve fibers in mesenteric blood vessels of rats irradiated with cobalt-60 gamma rays.

The effects of 60Co gamma rays on calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers and neuropeptide Y (NPY)-immunoreactive nerve fibers were examined in mesenteric blood vessels of rats. Using a free-floating immunostaining streptavidin-biotin peroxidase complex method combined with a nickel-enhancement technique, we found that the distribution pattern of these two peptidergic nerve fibers in superior mesenteric arteries and superior mesenteric veins did not change, while the densities of CGRP-immunoreactive nerve fibers and NPY-immunoreactive nerve fibers in superior mesenteric arteries and veins varied with the time after irradiation. The results suggested that CGRP and NPY may be important in the development and elimination of radiation-induced injury.

Treatment of bilateral intrahepatic stones with high duct strictures through selective central hepatic resection.

Bilateral intrahepatic duct stones with strictures, more common in the Orient than elsewhere, are difficult to access surgically. The results of surgical procedures have not been generally successful and are attended by a high postoperative complication and recurrence rate. We report the treatment of 30 patients with intrahepatic calculi and high duct strictures by means of selective central hepatic resection without dissection of the major vessels at the hilum. Central hepatic resection provided satisfactory access to the primary and secondary confluences of the intrahepatic ducts, allowed removal of residual stones and ascariasis, and permitted correction of multiple strictures. Twenty-nine patients so treated were followed for a mean of 32 months after operation. No patients developed recurrent fever, biliary colic, or jaundice after the operation. The technique is therefore recommended as an effective alternative to extensive hepatic lobectomy in the treatment of the intrahepatic calculi with multiple strictures.

Failure of nonimmunogenic islet allografts to induce donor-specific immunological unresponsiveness [corrected].

Highly purified perinatal rat islets, isolated by a nonenzymic in vitro culture technique, have been successfully transplanted across complete MHC barriers without immunosuppression. Acceptance of these allogeneically transplanted islets is hypothesized to result from an absence of antigen presenting cells (APCs) within the islets. This study was designed to examine the effects of organ transplantation and cyclosporine (CsA) therapy on the development of immunological unresponsiveness in recipients receiving a graft of culture-isolated islets. Kidneys were successfully allotransplanted into unilaterally nephrectomized rats, across a complete MHC barrier (Rt1lv1 to Rt1u) using CsA therapy initiated on the day of transplantation (7.5 mg/kg, orally for 14 days). Remaining native kidneys were removed 14 days following renal allotransplantation. Limited mononuclear cell (MNC) infiltrates were observed in biopsies of renal allografts, taken 30 days posttransplant, but failure of the renal allograft was not observed. Animals bearing established renal allografts (n = 10) received allografts of approximately 200 highly purified perinatal islets (ACl, n = 5; F-344, n = 5), transplanted to the kidney subcapsule of the established renal allograft at least 30 days following renal allotransplantation (at least 16 days following termination of CsA). Islet allografts were not rejected, and, as expected, did not initiate rejection of the renal allograft. Similar results were observed in renal allograft recipients rendered diabetic by a single injection of streptozotocin (STZ, 65 mg/kg, n = 5) and receiving islet allografts of sufficient mass (approximately 1200-1400 islets) to reverse STZ-induced hyperglycemia. Further, neither islet nor renal allografts were rejected following challenge by 1 x 10(7) donor-strain dendritic cells (DCs).(ABSTRACT TRUNCATED AT 250 WORDS)

A VIP/PHI-containing pathway links urinary bladder and sacral spinal cord.

Nerve fibres containing VIP and the co-produced PHI are found in the dorsal horn and autonomic centres of the sacral spinal cord and in pelvic organs. We have investigated the origin of these nerve fibres and a possible peptide-containing pathway linking pelvic viscera with the spinal cord of the cat and rat using neurochemical and neurosurgical procedures, retrograde tracing and immunocytochemistry. Cell bodies were located in the dorsal root ganglia (after colchicine injection), pelvic ganglia and bladder wall. Capsaicin treatment induced a loss of VIP/PHI from the dorsal horn. Retrograde tracing from the bladder revealed True Blue labelled cells in the dorsal root ganglia (L6, S1), parasympathetic nuclei and pelvic ganglia. Labelled cells were sequentially immunostained for VIP/PHI which were numerous in pelvic ganglia and scattered and weak in dorsal root ganglia. Pelvic nerve section induced a decrease of VIP/PHI immunoreactivity from the spinal cord and no change or a minimal increase in immunoreactive nerve fibers of the bladder. Thus pelvic visceral afferents with cell bodies in the dorsal root ganglia are a significant source of VIP/PHI-containing fibres in the sacral dorsal horn.

Composition and central projections of the pudendal nerve in the rat investigated by combined peptide immunocytochemistry and retrograde fluorescent labelling.

The central distribution of the afferent and efferent projections of pelvic striated muscles, the pudendal and sciatic nerves, were systemically investigated in rats by retrograde tracing techniques combined with immunocytochemistry using antibodies to 9 neuropeptides. True Blue was injected into either the pelvic muscles, pudendal or sciatic nerves. Seven days later the spinal cord and dorsal root ganglia (L3-S2 levels) were processed for immunocytochemistry. Injection of tracer into the pelvic muscles labelled dorsomedial, ventral and dorsolateral motoneuron groups of the L6 segment and a few sensory neurons in the respective dorsal root ganglia. Pudendal nerve injection also labelled the same motoneuron groups, 50% of neurons of the retrodorsolateral column and numerous sensory neurons of the dorsal root ganglion. Concomitant labelling of pudendal and sciatic nerves with different fluorescent tracers revealed a small number of double-labelled cells in the dorsal root ganglia but only single-labelled cells in the retrodorsolateral nucleus. Enkephalin-, somatostatin- and neuropeptide Y-containing fibres were particularly abundant in and around dorsomedial and dorsolateral groups as well as the intermediolateral cell column. We conclude that in the rat (a) the pudendal nerve has motor, sensory and autonomic (parasympathetic) components in contrast to the sciatic which is primarily motor and somatosensory, (b) some afferents from these nerves exhibit pre-spinal convergence and (c) dorsomedial and dorsolateral motoneuron groups are homologous to Onuf's nucleus in man.

CGRP-immunoreactive nerves in the genitalia of the female rat originate from dorsal root ganglia T11-L3 and L6-S1: a combined immunocytochemical and retrograde tracing study.

The origin of the calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibres in the genital organs of the female rat was investigated by immunocytochemistry and retrograde tracing with the fluorescent dye True blue. The tracing results revealed that these tissues receive an afferent nerve supply from two separate groups of dorsal root ganglia: T11-L3 and L6-S1. In T11-L3 ganglia 66-86% of True blue-labelled neurones displayed CGRP immunoreactivity whilst 45-63% of labelled cells in L6-S1 ganglia contained the peptide. The results indicate that CGRP-containing dorsal root ganglion neurones form a major part of the afferent sensory nerve supply to the female rat genitalia.

Demonstration of paracervical ganglion origin for the vasoactive intestinal peptide-containing nerves of the rat uterus using retrograde tracing techniques combined with immunocytochemistry and denervation procedures.

The origin of the abundant vasoactive intestinal peptide (VIP)-immunoreactive nerves in the uterus has not been fully determined. In this study, a fluorescent dye, True Blue was injected into the uterus of rat and 6 days later, neuronal cell bodies of the paracervical ganglion were found to be labelled by this dye. Some of these labelled ganglion cells were also found to contain VIP immunoreactivity by immunocytochemistry. When the preganglionic pelvic and/or hypogastric nerves of rats were sectioned, the VIP-immunoreactive nerves in the uteri were not depleted, indicating that these nerves did not originate from the splanchnic ganglion, dorsal root ganglion or the spinal cord. Therefore it is concluded that VIP-immunoreactive nerves in the uterus originate from the paracervical ganglion.

Primary neuroendocrine differentiated mucinous adenocarcinoma of the vulva: case report and review of the literature.

Only a few cases on mucinous adenocarcinomas of the vulva have been reported. In this study, we present a case of a 75-year-old woman with a tumor in the left major labium. Because biopsy had shown formations of squamous cell carcinoma, radical vulvectomy with bilateral inguinal and femoral lymph node dissection were performed. At that time, histology was interpreted as small-cell, anaplastic carcinoma, with focal epidermoid differentiation. Postoperative radiation therapy was performed. Sixteen months after surgery, the patient presented with bilateral breast carcinomas. Histology showed a scirrhous carcinoma of the left and a medullary carcinoma of the right breast, but no lymph node metastases. Histochemical and immunohistochemical re-examination of the vulvar carcinoma now revealed a mucinous adenocarcinoma with neuroendocrine differentiation. The tumor expressed neuroendocrine markers such as chromogranin A and protein gene-product (PGP) 9.5, as well as peptides of the vasoactive intestinal polypeptide (VIP) family, and serotonin. Histochemical silver stains demonstrated Grimelius argyrophilia and Masson argentaffinity. Because of positive estrogen and progesterone receptor status of both breast cancers, postoperative Tamoxifen therapy was performed. The patient is still alive four years after vulvectomy.

Stimulation of insulin release in rats by Die-Huang-Wan, a herbal mixture used in Chinese traditional medicine.

Die-Huang-Wan is a herbal mixture widely used in Chinese traditional medicine to treat diabetic disorders. We have investigated the effect of Die-Huang-Wan on plasma glucose concentration in-vivo. Die-Huang-Wan was administered orally (5.0, 15.0 or 26.0 mg kg(-1)) to three rat models. Wistar rats were used as the normal animal model, rats with insulin-resistance (induced by the repeated thrice daily injection of human long-acting insulin) were used as the non-insulin-dependent diabetic model, and streptozotocin-induced diabetic rats were used as the insulin-dependent diabetic model. In normal rats, approximately 1 h after oral administration of Die-Huang-Wan the plasma glucose concentration decreased significantly in a dose-dependent manner, from 5 to 26.0 mg kg(-1). A similar effect was observed in rats with insulin-resistance. However, this effect was not observed in streptozotocin-induced diabetic rats, even at an oral dose of 26.0 mg kg(-1). These results suggested an insulin-dependent action, a view supported by the increase of plasma insulin-like immunoreactivity in normal rats receiving Die-Huang-Wan. The results indicated that Die-Huang-Wan had an ability to stimulate the secretion of insulin and this preparation seemed helpful in improving the diabetic condition, especially hyperglycaemia in type-II diabetes.

Application of silver acetate autometallography and gold-silver staining methods for in situ DNA hybridization.

In situ hybridization using biotinylated DNA probes has become an important tool in histopathology. It is well known that the sensitivity of the methods used to demonstrate viral DNA in formalin-fixed and paraffin-embedded specimen depends strongly on the detection system used. In the present study, an optimized in situ DNA hybridization protocol was combined with four different approaches of gold-silver staining methods. For silver enhancement, the recently described method of silver acetate autometallography, a technique allowing highly efficient development without the necessity of dark room illumination has been used. The most efficient detection method found in our experiments was the use of gold-adsorbed anti-biotin antibodies with subsequent silver enhancement. This staining procedure can be completed in 5 hours including hybridization and is a highly sensitive alternative to peroxidase and alkaline phosphatase detection systems.

Serotypes, biotypes and antibiotic susceptibility of 126 clinical isolates of Haemophilus influenzae.

Serotypes, biotypes, and antibiotic susceptibility of 126 Haemophilus influenzae isolates were determined. Five of the 126 isolates were from blood and were encapsulated type b strains; those taken from other sites were not typable. There were 13% biotype I, 36% biotype II, 38% biotype III, 5% biotype IV, 4% biotype V, and 4% biotype VI isolates. Antibiotic susceptibility tests using the standard disk diffusion method showed the following resistance: ampicillin 51%, cefamandole 10%, cefuroxime 3%, chloramphenicol 28%, tetracycline 37% and sulfamethoxazole-trimethoprim 49%. None of the five type b isolates were resistant to cefotaxime, a third generation cephalosporin. The second generation cephalosporins, cefamandole and cefuroxime, showed a superior activity against H. influenzae isolates, compared to other antibiotics. Multiple drug resistance was found in 64 (51%) isolates. Four of the five type b isolates were resistant to multiple drugs. The multiple-resistance pattern most frequently observed was to ampicillin, chloramphenicol, tetracycline and sulfamethoxazole-trimethoprim. Most clinical isolates did not contain plasmids; therefore, the antibiotic resistance of these H. influenzae strains was probably chromosome-mediated.

Case study: how to apply data mining techniques in a healthcare data warehouse.

Healthcare provider organizations are faced with a rising number of financial pressures. Both administrators and physicians need help analyzing large numbers of clinical and financial data when making decisions. To assist them, Rush-Presbyterian-St. Luke's Medical Center and Hitachi America, Ltd. (HAL), Inc., have partnered to build an enterprise data warehouse and perform a series of case study analyses. This article focuses on one analysis, which was performed by a team of physicians and computer science researchers, using a commercially available on-line analytical processing (OLAP) tool in conjunction with proprietary data mining techniques developed by HAL researchers. The initial objective of the analysis was to discover how to use data mining techniques to make business decisions that can influence cost, revenue, and operational efficiency while maintaining a high level of care. Another objective was to understand how to apply these techniques appropriately and to find a repeatable method for analyzing data and finding business insights. The process used to identify opportunities and effect changes is described.

Ministry of Health clinical practice guidelines: depression.

The Ministry of Health (MOH) have updated the clinical practice guidelines on Depression to provide doctors and patients in Singapore with evidence-based treatment for depression. This article reproduces the introduction and executive summary (with recommendations from the guidelines) from the MOH clinical practice guidelines on Depression, for the information of readers of the Singapore Medical Journal. Chapters and page numbers mentioned in the reproduced extract refer to the full text of the guidelines, which are available from the Ministry of Health website: http://www.moh.gov.sg/content/moh_web/home/Publications/guidelines/cpg/2012/depression.html. The recommendations should be used with reference to the full text of the guidelines. Following this article are multiple choice questions based on the full text of the guidelines.