Anorectal Malformations and Associated Anomalies in Children.

Anorectal malformations (ARM) incorporate a broad spectrum of diseases, can affect both sexes, and involve the distal anus and rectum as well as the uro-genital tracts. Defects range from the minor which can be treated easily with an excellent outcome, to those are complex and often associated with other anomalies are difficult to manage with poor functional prognosis. This study was done to observe the hospital incidence of Anorectal malformations, frequency of types, sex distribution and spectrum of associations with ARM. The effects of presence of associated anomalies on morbidity and mortality also observed. Detailed history, clinical examinations and relevant investigations were performed for the primary and as well as the associated anomalies. A total of 80 patients were admitted in the department of pediatric surgery in Mymensingh Medical College Hospital during the period of June 2016 to May 2017. Age of the patients was ranging from 1-180 days with the mean age of 0.49±1.002 months. Male: Female ratio was 1.6: 1. Among them 48(60%) were high and 32(40%) were low variety of ARM. In male 37(46.2%) had high and 13(13.7%) were low variety whereas in female 11(13.7%) were high and 19(23.7%) had low ARM. Associated anomalies were seen in 25(31.2%) patient -18 in males and 7 in females; 20 in high and 5 in low ARM. Associated anomalies were uro-genital 11(13.8%), cardiovascular 10(12.5%), vertebral 4(5%), limb defects 3(3.5%) and others 2(2.5%). Four patients have more than one anomaly. Anorectal malformations occur more in boys than girls. Males were more likely to have high lesions and without fistula was the common defect. Low variety ARM were found more in females with Anovestibular fistula is the commonest defect. The most common associated anomalies were recto urinary fistula (13.8%). Associations were more in high than low ARM but not significant (p>0.05). Post operative complications were more in high ARM in both sexes with associated anomalies. The effects of types and associations on morbidity and mortality were significantly different (p<0.05).

Pesticides and their degradates in groundwater reflect past use and current management strategies, Long Island, New York, USA.

Long Island, New York, has a mix of urban/suburban to agricultural/horticultural land use and nearly 3 million residents that rely on a sole-source aquifer for drinking water. The analysis of shallow groundwater (<40 m below land surface) collected from 54 monitoring wells across Long Island detected 53 pesticides or pesticide degradates. Maximum concentrations for individual pesticides or pesticide degradates ranged from 3 to 368,000 ng/L. The highest concentrations and most frequent pesticide detections occurred in samples collected from the pesticide management (PM) network, set in an agricultural/horticultural area in eastern Long Island with coordinated pesticide management by state and local agencies. The other two networks (Suffolk and Nassau/Queens) were set in suburban and urban areas, respectively, and had less frequent detections and lower pesticide concentrations than the PM network. Pesticide detections and concentration patterns (herbicide, insecticide, or fungicide) differed among the three networks revealing broad differences in land use. The predominance of fungicides metalaxyl, 1H-1,2,4-triazole (propiconazole/myclobutanil degradate), and 4-hydroxychlorothalonil (HCTL, chlorothalonil degradate) in samples from the PM network reflects their intensive use in agricultural settings. Total fungicide concentrations in the PM network ranged from <10 to >300,000 ng/L. The widespread detection of imidacloprid and triazine herbicides, simazine and atrazine, reveal a mixture of current and past use pesticides across the Long Island region. Low concentrations (<200 ng/L) of the triazines in the Suffolk and Nassau/Queens networks may reflect a change in land use and application. Acetanilide herbicides and aldicarb have been discontinued for 20 and 40 years, respectively, yet the concentrations of their degradates were among the highest observed in this study. Acetanilide (total concentrations up to 10,000 ng/L) and aldicarb degradates (up to 270 ng/L) are present in the PM network at much lower concentrations than previous Long Island studies and reflect changes in agricultural practices and pesticide management.

Immune checkpoint expression on peripheral cytotoxic lymphocytes in cervical cancer patients: moving beyond the PD-1/PD-L1 axis.

Immune checkpoint therapy to reverse natural killer (NK) and T cell exhaustion has emerged as a promising treatment in various cancers. While anti-programmed cell death 1 (PD-1) pembrolizumab has recently gained Food and Drug Administration (FDA) approval for use in recurrent or metastatic cervical cancer, other checkpoint molecules, such as T cell immunoreceptor with immunoglobulin (Ig) and immunoreceptor tyrosine-based inhibition motif (ITIM) domains (TIGIT) and T cell immunoglobulin and mucin-domain containing-3 (Tim-3), have yet to be fully explored in this disease. We report expression of TIGIT, Tim-3 and PD-1 on subsets of peripheral blood NK (CD56dim/neg CD16bright/dim/neg and CD56bright CD16dim/neg ) and T cells. The percentages of these cells were increased in women with cervical cancer and pre-malignant lesions. PD-1+ NK and T cells were likely to co-express TIGIT and/or Tim-3. These cells, with an apparently 'exhausted' phenotype, were augmented in patients. A subset of cells were also natural killer group 2 member D (NKG2D)- and DNAX accessory molecule 1 (DNAM-1)-positive. PD-1int and PD-1high T cells were notably increased in cervical cancer. Soluble programmed cell death ligand 1 (PD-L1) was higher in cancer patient blood versus healthy donors and we observed a positive correlation between sPD-L1 and PD-1+ T cells in women with low-grade lesions. Within the cancer group, there were no significant correlations between sPD-L1 levels and cervical cancer stage. However, when comparing cancer versus healthy donors, we observed an inverse association between sPD-L1 and total T cells and a correlation between sPD-L1 and CD56dim NK cells. Our results may show an overview of the immune response towards pre-cancerous lesions and cervical cancer, perhaps giving an early clue as to whom to administer blocking therapies. The increase of multiple checkpoint markers may aid in identifying patients uniquely responsive to combined antibody therapies.

Evaluation of Breast Lump - Comparison between True-cut Needle Biopsy and FNAC in MMCH: A Study of 100 Cases.

One-fourth of all women suffer from breast disease in their lifetime. World Health Organization estimated that over 508,000 women died in 2011 due to breast cancer worldwide.For several years, fine needle aspiration cytology (FNAC) was the most practiced method for the pathological diagnosis of breast lump specially differentiation of benign from malignant. The advent of core needle or True-Cut biopsy (TCB) in the new millennium has resulted in many surgeons switching to TCB since it provides a sufficient amount of tissue for pathologists to make an accurate histological diagnosis.During the study period, patients present with clinically palpable breast lump admitted in different surgicalunits of MMCH, among them 100 patients selected purposively. Then a prospective comparative study was carried out in the Department of Surgery, Mymensingh Medical College Hospital, Mymensingh, Bangladesh from September 2017 to February 2018. Out of a total of 100 patients, who presented with suspicious breast lump, as clinically diagnosed 68 patients had benign breast lump and 32 patients had malignant breast lump. FNAC confirmed the diagnosis of breast carcinoma in 27 patients with sensitivity 89.65% and specificity 66.66%. True-cut biopsy confirmed the diagnosis of breast carcinoma in 29 patients with sensitivity 96.66% and specificity 100%. It also gave the definitive histological type and grade which correlated with the final histopathology report in 29 out of the 30 patientsTCB also provides adequate tissue for the evaluation of molecular markers which have extreme therapeutic value.

Motor Outcome of Stroke Patients with Low Dose Levodopa Therapy.

Stroke is one of the leading cause of disability worldwide. Motor function deficits due to stroke contribute to overall low quality of life. The objective was of this study is to observe functional motor outcome after stroke with low dose Levodopa therapy. This prospective follow up study was carried out in the Department of Neurology, Mymensingh Medical College Hospital, Mymensingh, Bangladesh from July 2014 to June 2016 to see the effect of low dose of Levodopa (110mg) on motor outcome after stoke disability. Motor deficit was measured by Medical Research Council (MRC) grading and Rivermead Mobility Index (RMI) score. Two groups were selected by simple random method, consisted of both ischemic and hemorrhagic stroke. All the patients of both the groups were suffering from at least some post stroke motor disability and attended full course of physiotherapy. The group (L) received 110mg Levodopa with physiotherapy. On the other hand (NL) group received only physiotherapy. They were all followed up for four times within two months of time and were assessed for recovery of motor function. Mean age was 59.03±11.56 years in Levodopa (L) group and 57.10±12.41 years in the Non Levodopa (NL) group; Males were predominant in both groups. Ninety three (77.50%) cases had ischemic stroke and 27(22.50%) cases had hemorrhagic stroke. Most common risk factors were hypertension and smoking. No known risk factor was detected in 8 (6.67%) patients. Single or multiple risk factors were confirmed in 112 patients (93.33%). MRC score was significantly higher both in affected upper and lower limb in Levodopa group comparing non Levodopa group at 4th visit. RMI score was also significantly higher in Levodopa group comparing non Levodopa group at 4th visit. The Levodopa (L) group showed better recovery pattern than Non Levodopa (NL) group. It can be concluded that motor recovery was better with administration of a single low dose of Levodopa in combination with physiotherapy. Motor outcome was significantly higher in levodopa group than non-levodopa group.

Incidence of Negative Appendicectomy in Clinically Diagnosed Patients of Acute Appendicitis.

This study was conducted in the department of Surgery, Mymensingh Medical College Hospital, Mymensingh, Bangladesh from January 2016 to December 2017 and performed to assess the negative appendicectomy on histopathology followed by clinically and laboratory investigations based diagnosed acute appendicitis. To perform this prospective study 200 patients with pain in right iliac fossa of both sexes randomly selected and were evaluated by history, clinical examination, laboratory investigations (USG, CBC) and scoring system. Among 200 patients male 124 and female 76 under went appendicectomy 36 were histopathologically normal appendix. Over all negative appendicectomy (NAR) 18%, among them 14.5% (18/124) were male & 23.67% (18/76) were female.

Association of Hypertension with Serum Estrogen Level in Postmenopausal Women.

Hypertension is a chronic and debilitating disease. Its complications give rise to cardiovascular diseases, stroke in Postmenopausal women. Estrogen deficiency that develops during menopause is likely the etiological factor for development of hypertension in postmenopausal women. Increased incidence of cardiovascular diseases in postmenopausal women may be due to hypertension caused by lower level of estrogen hormone. The study was carried out to observe the association of hypertension with serum estrogen level in postmenopausal women. This cross sectional study was conducted in the Department of Physiology, Dhaka Medical College, Dhaka, during the period of January 2011 to December 2011. A total number of 90 female subjects were selected from different areas of Dhaka city. Among them, 60 postmenopausal women with age ranging from 50 to 60 years were taken as study group and 30 apparently healthy premenopausal women with age ranging from 20 to 30 years were included as control group for comparison. Systolic blood pressure and diastolic blood pressure were recorded in both groups. Serum estrogen level was estimated in order to assess the hormonal level of both groups. Data was analyzed by Unpaired Student's 't' test and Pearson's co-efficient (r) test as applicable. The value of systolic blood pressure was higher in postmenopausal women than those of premenopausal women and result was statistically significant. The level of diastolic blood pressure was also significantly (p<0.001) higher in postmenopausal women in comparison to those of premenopausal women. In postmenopausal women serum estrogen level was lower than premenopausal women and serum estrogen level showed negative correlation with systolic and diastolic blood pressure levels. All these correlation were statistically non significant. Present study revealed that there is association of hypertension with serum estrogen level in postmenopausal women.

Preparation, nutritional composition, functional properties and antioxidant activities of multigrain composite mixes.

Multigrain composite mixes were prepared from different cereals, legumes, millets, nuts along with condiments by different processes. Multigrain composite mixes had 10 to 12% moisture, 56 to 61% carbohydrate, 15 to 20% protein, 9 to 13% crude lipid and 2 to 3% ash. Energy value ranged from ~1600 to 1700 kJ/100 g. Among the vitamins studied, thiamine and riboflavin content varied from 0. 23 to 0.45 mg% and from 8.7 to 21.6 microgram% respectively. Dietary fibre was in the range of 12.4-16.5%. Swelling power of these mixes was about 10; however solubility varied from 17 to 22%. In-vitro Starch digestibility varied from 60 to 76%. Phytic acid content in these multigrain composite mixes varied from 0.6 to 0.8%. Poly-phenols ranged from 1.2 to 1.5%, DPPH free radical scavenging activity ranged from 75.2-86.2% and metal chelating activity ranged from 1.9 to 3.9%. Pasting profile by a Brabender Viscograph of these mixes indicated that they have cross linked starch type behaviour. These multigrain composite mixes can be used for the preparation of food formulations, savory products, pan cake, snacks preparation like muruku and chakli.

Effect of vitamins C and E on antioxidant status of breast-cancer patients undergoing chemotherapy.

WHAT IS KNOWN AND OBJECTIVE: Reactive oxygen/nitrogen species generated by antineoplastic agents are prime suspects for the toxic side-effects of acute or chronic chemotherapy. The present study was undertaken to test whether vitamins C and E (VCE) supplementation protect against some of the harmful effects of commonly used anticancer drugs in breast-cancer patients. METHODS: In a randomized 5-month study, the activity of various antioxidant enzymes (superoxide dismutase, catalase, glutathione-S-transferase and glutathione reductase) and the levels of malondialdehyde and reduced glutathione were measured in forty untreated breast-cancer patients (stage II) and compared with those of healthy controls. The degree of DNA damage was also assessed in the peripheral lymphocytes of the patients by alkaline single cell gel electrophoresis. The untreated patients were then randomly assigned to either treatment with chemotherapy alone (5-fluorouracil 500 mg/m(2) i.v. day 1, doxorubicin 50 mg/m(2) i.v. day 1 and cyclophosphamide 500 mg/m(2) i.v. day 1, every 3 weeks for six cycles) or to the same chemotherapy regimen supplemented with VCE (vitamin C 500 mg tablet and vitamin E 400 mg gelatin capsule). On completion of the treatments, both the groups were studied again for the levels of the markers measured prior to treatment. RESULTS AND DISCUSSION: The untreated group showed significantly lower levels of antioxidant enzymes (P < 0·001) and reduced glutathione (P < 0·001), and more extensive lipid peroxidation (P < 0·001) and DNA damage than healthy controls. Similar but less pronounced patterns were observed in the patients receiving chemotherapy alone. The group of patients receiving VCE supplementation had all the marker levels moving towards normal values. Activities of superoxide dismutase, catalase, glutathione-S-transferase and glutathione reductase, and the levels of reduced glutathione were significantly increased (P < 0·01) while, the levels of malondialdehyde and DNA damage were significantly (P < 0·01) reduced in the VCE supplemented group relative to those of patients receiving chemotherapy alone as well as relative to the pretreatment levels. WHAT IS NEW AND CONCLUSION: Co-administration of VCE restored antioxidant status, lowered by the presence of breast-cancer and chemotherapy. DNA damage was also reduced by VCE. The results suggest that VCE should be useful in protecting against chemotherapy-related side-effects and a randomized control trial to evaluate the effectiveness of VCE in breast-cancer patients using clinical outcomes would be appropriate.

The actin-bundling protein fascin is overexpressed in colorectal adenomas and promotes motility in adenoma cells in vitro.

BACKGROUND: Fascin is overexpressed in many cancers, including colorectal, but its role in the malignant transformation of benign colorectal adenomas is unclear. METHODS: Immunohistochemical analysis of fascin expression was carried out in resected human colorectal adenoma specimens. The effects of forced overexpression of fascin on adenoma cell motility were also analysed. RESULTS: We show fascin overexpression in adenomas increasing with tumour size, histological type, and degree of dysplasia and increased cell motility in adenoma cell lines following fascin transfection. CONCLUSION: These data suggest an important role for fascin in the malignant progression of colorectal tumours.

Inhibition of COX-2 with NS-398 decreases colon cancer cell motility through blocking epidermal growth factor receptor transactivation: possibilities for combination therapy.

UNLABELLED: The use of non-steroidal anti-inflammatory drugs has proved of great interest in the prevention and treatment of colorectal cancer, although their precise mechanisms of action remain unclear. Overexpression of cyclooxygenase-2 (COX-2) and subsequent prostaglandin production promote metastasis and have been shown to increase cell motility in vitro. OBJECTIVE: We have aimed to elucidate whether specific inhibition of COX-2 with NS-398 (NS-398 is a selective inhibitor of COX-2) would be able to inhibit motility of colorectal cancer cells and whether this was modulated through epidermal growth factor receptor (EGFR) transactivation. MATERIALS AND METHODS: A transwell filter assay was used to study cell motility. Expression of COX-2, EGFR phosphorylation and prostaglandin E(2) (PGE(2)) receptors were assessed by Western blot analysis and reverse transcriptase-polymerase chain reaction. PGE(2) concentrations after NS-398 treatment were estimated by enzyme immunoassay. RESULTS: Treatment with NS-398 significantly reduced PGE(2) levels and reduced cell migration in the HT29 and HCA7 colorectal carcinoma cell lines and this effect was rescued by addition of PGE(2). Furthermore, specific inhibition of COX-2 with NS-398 reduced EGFR phosphorylation in colorectal cancer cells. Direct inhibition of EGFR activity with AG1478 reduced PGE(2)-stimulated motility, clearly demonstrating that PGE(2 )acts via the EGFR-signalling pathway. The novel combination of NS-398 and AG1478 dramatically reduced migration of colorectal cancer cells. CONCLUSION: The data presented indicate that the use of NS-398 in chemoprevention and adjuvant therapy for colorectal cancer may work in part, through the inhibition of cell motility. Furthermore, our data suggest that the combined use of non-steroidal anti-inflammatory drugs with EGFR antagonists could be explored further for future use in the clinic.

Camptomelic dysplasia with sex reversal.

Camptomelic dysplasia is a disorder of the newborn characterized by congenital bowing and angulation of long bones together with other skeletal and extraskeletal defects. The affected newborn had dysmorphic features with bowing of the legs and bilateral talipes equinovarus. Radiology showed marked anterior bowing of both tibia with disproportionately short fibula, anterolateral bowing of the femurs and wide pelvic outlet with small iliac wings. She had sex reversal with normal female genitalia and 46, XY karyotype. Camptomelic dysplasia is generally considered to be a lethal skeletal dysplasia and most patients die in the neonatal period due to severe respiratory distress. Survivors may have learning difficulties, developmental delay, conductive hearing loss, myopia and recurrent chest infections. Because of its high associated mortality, prenatal diagnosis of camptomelic dysplasia is mandatory. The birth of a child with skeletal dysplasia is an emotionally difficult experience for parents.

Cytokine-augmented culture of haematopoietic progenitor cells in a novel three-dimensional cell growth matrix.

Studies aimed at the in vitro expansion of haematopoietic progenitor cells (HPCs) have suffered from the conflict of increasing cell numbers while maintaining long-term repopulating ability. We have developed a long-term bone marrow bioreactor culture system resembling the marrow-microenvironment that cultures HPCs in an inert, three-dimensional, porous biomatrix termed Cellfoam. Previous studies have shown that the short-term culture of CD34(+)cells in Cellfoam improved the maintenance and multipotency of haematopoietic stem cells compared to cells cultured on plastic dishes. In this study, we examined the effects of low concentrations of cytokines including stem cell factor (SCF), IL-3, and Flk-2/Flt-3 ligand, on the maintenance, preservation and multipotency of CD34(+) cells cultured for 3 or 6 weeks in Cellfoam. Analysis of cell yields using flow cytometry showed that in SCF and Flk-2/Flt-3 ligand-supplemented cultures as well as cytokine-free cultures, a higher number of CD45(+)34(+) and CD45(+)34(+)38(-) cells is observed in Cellfoam cultures as compared to plastic cultures. The function of cultured cells was evaluated in colony-forming assays. The data demonstrate that Cellfoam cultures supplemented with SCF and Flk-2/Flt-3 ligand resulted in a higher output of colony activity compared to plastic cultures. Analysis of CAFC (29 days) activity also demonstrated that primitive progenitors were maintained to a greater extent in Cellfoam cultures containing either no cytokines or low concentrations of early-acting cytokines. These data suggest that culture of HPCs in three-dimensional bioreactors such as Cellfoam for extended periods may benefit from the addition of low levels of early-acting cytokines, including SCF and Flk-2/Flt-3 ligand, resulting in high yields of cells that are enriched for multipotent haematopoietic progenitors. These findings demonstrate that a three-dimensional matrix promotes the survival of primitive HPCs in culture and may modulate the in vitro effects of cytokines.

Inflammation and glandular proliferation in hyperplastic prostates: association with prostate specific antigen value.

A series of 73 cases of surgically resected prostatic tissue were histologically diagnosed as nodular hyperplasia, of which 10 (13.69%) cases had chronic prostatitis. The mean value of prostate specific antigen in prostatic hyperplasia without chronic prostatitis and prostatic hyperplasia with chronic prostatitis were 6.09 ng/ml and 13.61 ng/ml respectively. A statistically significant difference of prostate specific antigen level between these two groups were noted (P<.05). The degree of glandular proliferation in nodular hyperplasia was subjectively assessed and was recorded as mild, moderate or severe degree of proliferation. The mean value of prostate specific antigen were 3.28 ng/ml in patients with mild proliferation, 7.21 ng/ml in those with moderate proliferation and 14.78 ng/ml in those with severe proliferation. A significant association between prostate specific antigen level and degree of glandular proliferation was found (P<.05). Chronic prostatitis and glandular proliferation are the two important factors contributing to serum prostate specific antigen elevation in hyperplastic prostates.

Correlation of ultrasonographic findings with ultrasound guided fine needle aspiration of liver lesion.

Ultrasonographic findings of liver were correlated with cytological findings in a series of 50 patients. Multiple lesions described by ultrasonography and suggested as HCC/TB were proved to be metastatic in 60% cases and hepatocellular carcinoma in 40% cases by cytological examination. Multiple lesions suggested as metastatic lesions in ultrasonography was proved as such by cytology in 83% cases. Solitary lesion suggested as neoplastic in ultrasonography was proved as such in cytology in 90% cases. Of the 2 patients suggested as diffuse parenchymal lesion revealed cytological findings of cirrhosis in one case and that of TB in other. Serum alpha-feto protein and Carcinoembryogenic antigen (CEA) was done in all the cases. Serum alpha-feto protein was higher in hepatocellular carcinoma and CEA was higher in metastatic lesions. Ultrasound guided fine needle aspiration of liver can play more role in diagnosis and classification of liver disease than ultrasonographic comment alone, as it requires greater degree of precision to reach diagnostic accuracy.

TGF-beta1 down-regulates induced expression of both class II MHC and B7-1 on primary murine renal tubular epithelial cells.

BACKGROUND: We examined the immunomodulatory effects of transforming growth factor-beta1 (TGF-beta1) on the regulation of class II MHC and costimulatory molecule expression in a primary renal tubular epithelial cell line, called F1K. METHODS: Class II major histocompatibility complex (MHC), class II transactivator, B7-1, intercellular adhesion molecule-1 (ICAM-1) and interferon-gamma (IFN-gamma) receptor beta chain were evaluated in untreated and cytokine-treated F1K by Northern hybridization analysis and flow cytometry. T cell activation studies were performed to assess TGF-beta1-mediated effects on antigen presenting cell function of F1K. RESULTS: Pretreatment of F1K with TGF-beta1 markedly inhibited IFN-gamma-induced class II MHC expression, by both FACS and Northern analysis. Total class II transactivator mRNA levels were also diminished by TGF-beta1, indicating that class II MHC modulation in F1K results from inhibition of this intermediate protein. As previous studies demonstrated that cotreatment of F1K cells with IFN-gamma + lipopolysaccharide (LPS) induces B7-1, we evaluated the potential regulatory effects of TGF-beta1 exposure on B7-1 expression. Our studies revealed that B7-1 mRNA and cell-surface expression in IFN-gamma + LPS-treated F1K were decreased by TGF-beta1 pretreatment. Functional studies evaluating TGF-beta1-mediated effects were performed with IFN-gamma + LPS-treated F1K and MR1.3, a nephritogenic CD4+ Th2 clone derived from kidneys of animals with autoimmune glomerulonephritis. Interleukin (IL)-4 production assays demonstrated activation of MR1. 3 by IFN-gamma + LPS-treated cells, but not by IFN-gamma + LPS-treated cells previously exposed to TGF-beta1, indicating that TGF-beta1-mediated inhibition of class II MHC and B7-1 expression alters the antigen presenting cell function of F1K. CONCLUSIONS: These studies describe the proscriptive influence of TGF-beta1 on class II MHC and B7-1 expression in renal tubular epithelial cells. Such findings indicate that TGF-beta1 alters the antigen presenting cell function of renal tubular epithelial cells in vitro, and suggest a potential mechanism for immunosuppression of T cell-mediated renal immune responses in vivo.

Modulation of haematopoietic progenitor development by FLT-3 ligand.

The Flt-3 receptor is expressed in primitive haematopoietic cells and its ligand exerts proliferative effects on these cells in vitro in synergy with other cytokines. To increase our knowledge of the functional properties of the human Flt-3 ligand (FL) as relating to in vitro expansion of haematopoietic stem cells, the effects on murine haematopoiesis of FL alone or in combination with other growth factors were studied. Analysis of Flk-2/Flt-3 mRNA expression indicated that Flk-2/Flt-3 was preferentially expressed in primitive haematopoietic cell populations. To examine the expression of the Flk-2/Flt-3 receptor on megakaryocyte progenitors (CFU-Meg), Flk-2/Flt-3 positive and negative CD34(+)populations were separated from human bone marrow and cultured in a plasma clot culture system. CFU-Meg colonies were found in the Flk-2/Flt-3 negative fraction. Myeloid (CFU-GM) derived colonies appeared in the presence of FL alone. Neither FL+IL-3 nor FL+IL-3+IL-6 had any effect on the generation of megakaryocyte colonies (CFU-MK), due to the lack of FL receptor expression on megakaryocyte progenitors. Bone marrow cells remaining after 5-fluorouracil (5-FU) treatment of mice represent a very primitive population of progenitors enriched for reconstituting stem cells. This cell population expressed FL receptors, as revealed by RT-PCR analysis. Addition of FL alone did not enhance the replication of such cells in liquid cultures as compared to controls. However, a significantly greater generation of myeloid progenitors (CFU-GM) in clonogenic assays was observed in the presence of FL+IL-3, FL+GM-CSF or FL+CSF-1. In addition, the effects of FL on in vitro expansion of murine haematopoietic stem cells were studied using lineage-negative (lin(-)) Sca-1 positive (Sca-1(+)) c-kit positive (c-kit(+)) marrow cells from 5-FU treated mice. FL enhanced the survival of primitive murine lin(-)Sca-1(+)c-kit(+)cells. FL and IL-6 were able to significantly expand murine progenitor stem cells in vitro and promote their survival. These studies strongly suggest that FL significantly and selectively enhanced the generation of myeloid progenitors in vitro and increased myeloid progenitor responsiveness to later acting growth factors. In addition, FL synergized with IL-6 to support in vitro expansion of haematopoietic progenitors and promoted the survival of lin(-)Sca-1(+)c-kit(+)cells.

Immune reactivity following CD40L blockade: role in autoimmune glomerulonephritis in susceptible recipients.

To investigate the role of costimulation in autoimmune glomerulonephritis that develops in the setting of murine chronic graft-vs-host disease (cGVHD), we examined the effects of blocking CD40L, a costimulatory marker expressed on activated CD4+ T cells, in recipient mice. These studies addressed the potential role of CD40L blockade in preventing disease and in downregulating its expression in animals with evidence of autoreactivity. Animals treated acutely with anti-CD40L antibody at disease induction do not develop circulating anti-DNA antibodies, proteinuria, or histologic evidence of renal disease. If treatment is delayed for two weeks, after circulating anti-DNA antibodies are apparent, all animals develop massive proteinuria by 14 weeks after disease induction. Renal histology of kidneys from the delayed treatment and control groups reveal similar glomerular immune deposits, and intense staining for CD4, ICAM-1, and I-A(b) in areas of mononuclear cell infiltration. Long-term treatment studies begun two weeks after disease induction is not disease-protective, as all animals develop massive proteinuria and renal disease by 14 weeks. These studies suggest that early CD40L signaling events are critical to induction of allogeneic interactions and autoreactivity in cGVHD, but that short-term or chronic CD40L blockade, once autoreactivity is evident, does not abrogate systemic autoreactivity and renal involvement.

IFN-gamma and LPS differentially modulate class II MHC and B7-1 expression on murine renal tubular epithelial cells.

BACKGROUND: We have investigated inducible class II major histocompatibility complex (MHC) and B7 expression on primary murine renal tubular epithelial cells, called F1K cells, and examined their role in activating nephritogenic T cells derived from kidneys of animals with autoimmune glomerulonephritis. METHODS: Class II MHC, class II transactivator, and costimulatory molecule expression were evaluated in untreated and cytokine-treated F1K cells by Northern hybridization and flow cytometry. Cell-surface B7-1 expression was evaluated in vitro by immunoprecipitation and in vivo by immunohistochemistry. T-cell activation studies were then performed to assess the functional significance of B7-1 expression on F1K cells. RESULTS: Coincubation of F1K cells with interferon (IFN)-gamma and lipopolysaccharide (LPS) significantly decreased IFN-gamma-induced class II MHC expression, by both fluorescence-activated cell sorting and Northern analyses. LPS-mediated inhibition of class II MHC in this setting was effected through a decrease in class II transactivator mRNA levels in treated F1K cells. By contrast, IFN-gamma and LPS coincubation induced B7-1 but not B7-2 expression in F1K cells, as detected by Northern analysis, flow cytometry, and immunoprecipitation. In addition, renal tubular staining for B7-1 was apparent in kidneys isolated from IFN-gamma+LPS-treated recipient mice, as well as mice with autoimmune glomerulonephritis. Further studies evaluated the interaction of F1K cells and MR1.3, a nephritogenic CD4+ Th2 clone derived from kidneys of animals with autoimmune glomerulonephritis. Cytokine production assays revealed that F1K cells activated MR1.3 cells if they were pretreated with both IFN-gamma and LPS 48 hours prior to exposure to nephritogenic T cells. CONCLUSIONS: These studies are the first description of a differential regulation of class II MHC and B7-1 expression in renal tubular epithelial cells mediated by IFN-gamma and LPS. Such findings indicate that discrete proinflammatory stimuli could potentiate antigen-presenting capabilities of renal tubular epithelial cells in vivo and further suggest a direct role of such nonprofessional antigen-presenting cells in modulating renal immune responses.

Recombinant adeno-associated virus-based vectors provide short-term rather than long-term transduction of primitive hematopoietic stem cells.

Bone marrow stem cells collected from B6-Gpi-1a mice pretreated with 5-fluorouracil were incubated for 2 h at 37 degrees C in the presence of the recombinant adenovirus-associated virus-based vector (rAAV) SSV9. As measured in vitro immediately following transduction, SSV9 was found to be effective in transducing the primitive cobble-stone-area-forming cell (CAFC)-35 subset (60% transduction efficiency). However, this did not predict long-term expression as the presence of the transgene could not be detected six months after transplantation of 1-2 x 106 transduced bone marrow stem cells into lethally irradiated recipients. CAFC analysis of bone marrow cells and Southern blot analysis of bone marrow and spleen cells were negative, and polymerase chain reaction analysis showed less than 0.1% transduction in bone marrow cells. Therefore, based on our study we conclude that rAAV transiently transduces hematopoietic stem cells but fails to integrate into the genome, leading to the loss of the reporter gene within the first six months after transplantation in vivo.