Inhibition of Hippocampal Regeneration by Adjuvant Dexamethasone in Experimental Infant Rat Pneumococcal Meningitis

Pneumococcal meningitis (PM) causes neurological sequelae in up to half of surviving patients. Neuronal damage associated with poor outcome is largely mediated by the inflammatory host response. Dexamethasone (DXM) is used as an adjuvant therapy in adult PM, but its efficacy in the treatment of pneumococcal meningitis in children is controversially discussed. While DXM has previously been shown to enhance hippocampal apoptosis in experimental PM, its impact on hippocampal cell proliferation is not known. This study investigated the impact of DXM on hippocampal proliferation in infant rat PM. Eleven-day-old nursing Wistar rats (n = 90) were intracisternally infected with Streptococcus pneumoniae to induce experimental meningitis. Treatment with DXM or vehicle was started 18 h after infection, concomitantly with antibiotics (ceftriaxone 100 mg/kg of body weight twice a day [b.i.d.]). Clinical parameters were monitored, and the amount of cells with proliferating activity was assessed using in vivo incorporation of bromodeoxyuridine (BrdU) and an in vitro neurosphere culture system at 3 and 4 d postinfection. DXM significantly worsened weight loss and survival. Density of BrdU-positive cells, as an index of cells with proliferating activity, was significantly lower in DXM-treated animals compared to vehicle controls (P < 0.0001). In parallel, DXM reduced neurosphere formation as an index for stem/progenitor cell density compared to vehicle treatment (P = 0.01). Our findings provide clear evidence that DXM exerts an antiproliferative effect on the hippocampus in infant rat PM. We conclude that an impairment of regenerative hippocampal capacity should be taken into account when considering adjuvant DXM in the therapeutic regimen for PM in children.

Improving chondrogenesis: potential and limitations of SOX9 gene transfer and mechanical stimulation for cartilage tissue engineering

Articular cartilage injuries and degeneration affect a large proportion of the population in developed countries world wide. Stem cells can be differentiated into chondrocytes by adding transforming growth factor-beta1 and dexamethasone to a pellet culture, which are unfeasible for tissue engineering purposes. We attempted to achieve stable chondrogenesis without any requirement for exogenous growth factors. Human mesenchymal stem cells were transduced with an adenoviral vector containing the SRY-related HMG-box gene 9 (SOX9), and were cultured in a three-dimensional (3D) hydrogel scaffold composite. As an additional treatment, mechanical stimulation was applied in a custom-made bioreactor. SOX9 increased the expression level of its known target genes, as well as its cofactors: the long form of SOX5 and SOX6. However, it was unable to increase the synthesis of sulfated glycosaminoglycans (GAGs). Mechanical stimulation slightly enhanced collagen type X and increased lubricin expression. The combination of SOX9 and mechanical load boosted GAG synthesis as shown by (35)S incorporation. GAG production rate corresponded well with the amount of (endogenous) transforming growth factor-beta1. Finally, cartilage oligomeric matrix protein expression was increased by both treatments. These findings provide insight into the mechanotransduction of mesenchymal stem cells and demonstrate the potential of a transcription factor in stem cell therapy.

Micromass co-culture of human articular chondrocytes and human bone marrow mesenchymal stem cells to investigate stable neocartilage tissue formation in vitro

Cell therapies for articular cartilage defects rely on expanded chondrocytes. Mesenchymal stem cells (MSC) represent an alternative cell source should their hypertrophic differentiation pathway be prevented. Possible cellular instruction between human articular chondrocytes (HAC) and human bone marrow MSC was investigated in micromass pellets. HAC and MSC were mixed in different percentages or incubated individually in pellets for 3 or 6 weeks with and without TGF-beta1 and dexamethasone (±T±D) as chondrogenic factors. Collagen II, collagen X and S100 protein expression were assessed using immunohistochemistry. Proteoglycan synthesis was evaluated applying the Bern score and quantified using dimethylmethylene blue dye binding assay. Alkaline phosphatase activity (ALP) was detected on cryosections and soluble ALP measured in pellet supernatants. HAC alone generated hyaline-like discs, while MSC formed spheroid pellets in ±T±D. Co-cultured pellets changed from disc to spheroid shape with decreasing number of HAC, and displayed random cell distribution. In -T-D, HAC expressed S100, produced GAG and collagen II, and formed lacunae, while MSC did not produce any cartilage-specific proteins. Based on GAG, collagen type II and S100 expression chondrogenic differentiation occurred in -T-D MSC co-cultures. However, quantitative experimental GAG and DNA values did not differ from predicted values, suggesting only HAC contribution to GAG production. MSC produced cartilage-specific matrix only in +T+D but underwent hypertrophy in all pellet cultures. In summary, influence of HAC on MSC was restricted to early signs of neochondrogenesis. However, MSC did not contribute to the proteoglycan deposition, and HAC could not prevent hypertrophy of MSC induced by chondrogenic stimuli.

Glucocorticoids protect renal mesangial cells from apoptosis by increasing cellular sphingosine-1-phosphate

Neutral ceramidase (NCDase) and sphingosine kinases (SphKs) are key enzymes regulating cellular sphingosine-1-phosphate (S1P) levels. In this study we found that stress factor-induced apoptosis of rat renal mesangial cells was significantly reduced by dexamethasone treatment. Concomitantly, dexamethasone increased cellular S1P levels, suggesting an activation of sphingolipid-metabolizing enzymes. The cell-protective effect of glucocorticoids was reversed by a SphK inhibitor, was completely absent in SphK1-deficient cells, and was associated with upregulated mRNA and protein expression of NCDase and SphK1. Additionally, in vivo experiments in mice showed that dexamethasone also upregulated SphK1 mRNA and activity, and NCDase protein expression in the kidney. Fragments (2285, 1724, and 1126 bp) of the rat NCDase promoter linked to a luciferase reporter were transfected into rat kidney fibroblasts and mesangial cells. There was enhanced NCDase promoter activity upon glucocorticoids treatment that was abolished by the glucocorticoid receptor antagonist RU-486. Single and double mutations of the two putative glucocorticoid response element sites within the promoter reduced the dexamethasone effect, suggesting that both glucocorticoid response elements are functionally active and required for induction. Our study shows that glucocorticoids exert a protective effect on stress-induced mesangial cell apoptosis in vitro and in vivo by upregulating NCDase and SphK1 expression and activity, resulting in enhanced levels of the protective lipid second messenger S1P.

TNFα inhibits the development of osteoclasts through osteoblast-derived GM-CSF

Inflammatory cytokines such as tumor necrosis factor-alpha (TNFα) are potent stimulators of osteoclast formation and bone resorption and are frequently associated with pathologic bone metabolism. The cytokine exerts specific effects on its target cells and constitutes a part of the cellular microenvironment. Previously, TNFα was demonstrated to inhibit the development of osteoclasts in vitro via an osteoblast-mediated pathway. In the present study, the molecular mechanisms of the inhibition of osteoclastogenesis were investigated in co-cultures of osteoblasts and bone marrow cells (BMC) and in cultures of macrophage-colony stimulating factor (M-CSF) dependent, non-adherent osteoclast progenitor cells (OPC) grown with M-CSF and receptor activator of NF-κB ligand (RANKL). Granulocyte-macrophage colony stimulating factor (GM-CSF), a known inhibitor of osteoclastogenesis was found to be induced in osteoblasts treated with TNFα and the secreted protein accumulated in the supernatant. Dexamethasone (Dex), an anti-inflammatory steroid, caused a decrease in GM-CSF expression, leading to partial recovery of osteoclast formation. Flow cytometry analysis revealed that in cultures of OPC, supplemented with 10% conditioned medium (CM) from osteoblasts treated with TNFα/1,25(OH)(2)D(3), expression of RANK and CD11c was suppressed. The decrease in RANK expression may be explained by the finding, that GM-CSF and the CM from wt osteoblasts were found to suppress the expression of c-Fos, Fra-1, and Nfatc-1. The failure of OPC to develop into CD11c(+) dendritic cells suggests that cell development is not deviated to an alternative differentiation pathway, but rather, that the monocytes are maintained in an undifferentiated, F4/80(+), state. The data further implies possible interactions among inflammatory cytokines. GM-CSF induced by TNFα acts on early hematopoietic precursors, inhibiting osteoclastogenesis while acting as the growth factor for M-CSF independent inflammatory macrophages. These in turn may condition a microenvironment enhancing osteoclast differentiation and bone resorption upon migration of the OPC from circulation to the bone/bone marrow compartment.

Owner assessment in judging the efficacy of airway disease treatment

REASONS FOR PERFORMING STUDY: Efficacy of medications for recurrent airway obstruction is typically tested using clinical, cytological and lung function examinations of severely affected animals. These trials are technically challenging and may not adequately reflect the spectrum of disease and owner complaints encountered in clinical practice. OBJECTIVE: To determine if owners of horses with chronic airway disease are better able to detect drug efficacy than a veterinarian who clinically examines horses infrequently. METHOD: In a double-blinded randomised controlled trial, owners and a veterinarian compared the efficacy of dexamethasone (0.1 mg/kg bwt per os, q. 24 h, for 3 weeks; n = 9) to placebo (n = 8) in horses with chronic airway disease. Before and after treatment, owners scored performance, breathing effort, coughing and nasal discharge using a visual analogue scale (VAS). The clinician recorded vital parameters, respiratory distress, auscultation findings, cough and nasal discharge, airway mucus score, bronchoalveolar lavage fluid (BALF) cytology and arterial blood gases. RESULTS: The VAS score improved significantly in dexamethasone- but not placebo-treated horses. In contrast, the clinician failed to differentiate between dexamethasone- and placebo-treated animals based on clinical observations, BALF cytology or endoscopic mucus score. Respiratory rate (RR) and arterial oxygen pressure (PaO(2)) improved with dexamethasone but not placebo. CONCLUSIONS AND CLINICAL RELEVANCE: In the design of clinical trials of airway disease treatments, more emphasis should be placed on owner-assessed VAS than on clinical, cytological and endoscopic observations made during brief examinations by a veterinarian. Quantifiable indicators reflecting lung function such as RR and PaO(2) provide a good assessment of drug efficacy.

In vitro effect of different mediators of apoptosis on canine cranial and caudal cruciate ligament fibroblasts and its reversibility by pancaspase inhibitor zVAD.fmk

Primary fibroblast cultures of canine cranial (CCL) and caudal (CaCL) cruciate ligaments were stimulated with different apoptosis inducers with or without preincubation of the pancaspase inhibitor zVAD.fmk. In contrast to CaCL fibroblasts, fibroblasts from CCL were significantly more susceptible to apoptosis inducers of the intrinsic pathway like doxorubicin, cisplatin and nitric oxide (NO)-donors and to Fas ligand (FasL), an apoptosis inducer of the death receptor pathway. Apoptotic response to staurosporine and the peroxynitrite donor GEA was similar in both ligament fibroblasts. Stimulation with dexamethasone or TNFalpha could not induce apoptosis in CCL and CaCL fibroblasts, in spite of present TNFR1 and TNFR2 receptors. zVAD.fmk was able to prevent apoptosis in up to 66% of CCL cells when treated with FasL, cisplatin or doxorubicin but it had no effect on NO or peroxynitrite induced apoptosis. In conclusion, differential susceptibility to apoptotic triggers like FasL or NO between cranial and caudal cruciate ligament fibroblasts in vitro may be a reflection of the different susceptibilities to degenerative rupture of the ligament. These findings indicate that a general caspase inhibition does not completely protect canine CCL cells from apoptosis.

Acute Encephalopathy with Unilateral Cortical-Subcortical Lesions in Two Unrelated Kindreds Treated with Glucocorticoids Prenatally for Congenital Adrenal Hyperplasia due to 21-Hydroxylase Deficiency: Established Facts and Novel Insight

Background: Prenatal glucocorticoid (GC) treatment of the female fetus with 21-hydroxylase deficiency (21-OHD) may prevent genital virilization and androgen effects on the brain, but prenatal GC therapy is controversial because of possible adverse effects on fetal programming, the cardiovascular system and the brain. Case Reports: We report 2 patients with congenital adrenal hyperplasia (CAH) due to 21-OHD who were treated prenatally with dexamethasone, suffered from an acute encephalopathy and showed focal and multifocal cortical and subcortical diffusion restrictions in early MRI and signs of permanent alterations in the follow-up neuroimaging studies. Both patients recovered from the acute episode. Whereas the first patient recovered without neurological sequelae the second patient showed hemianopsia and spastic hemiplegia in the neurological follow-up examination. Conclusion: These are 2 children with CAH, both treated prenatally with high doses of dexamethasone to prevent virilization. The question arises whether prenatal high-dose GC treatment in patients with CAH might represent a risk factor for brain lesions in later life. Adverse effects/events should be reported systematically in patients undergoing prenatal GC treatment and long-term follow-up studies involving risk factors for cerebrovascular disease should be performed.

Isolated autosomal dominant growth hormone deficiency: stimulating mutant GH-1 gene expression drives GH-1 splice-site selection, cell proliferation, and apoptosis

The majority of mutations that cause isolated GH deficiency type II (IGHD II) affect splicing of GH-1 transcripts and produce a dominant-negative GH isoform lacking exon 3 resulting in a 17.5-kDa isoform, which further leads to disruption of the GH secretory pathway. A clinical variability in the severity of the IGHD II phenotype depending on the GH-1 gene alteration has been reported, and in vitro and transgenic animal data suggest that the onset and severity of the phenotype relates to the proportion of 17.5-kDa produced. The removal of GH in IGHD creates a positive feedback loop driving more GH expression, which may itself increase 17.5-kDa isoform productions from alternate splice sites in the mutated GH-1 allele. In this study, we aimed to test this idea by comparing the impact of stimulated expression by glucocorticoids on the production of different GH isoforms from wild-type (wt) and mutant GH-1 genes, relying on the glucocorticoid regulatory element within intron 1 in the GH-1 gene. AtT-20 cells were transfected with wt-GH or mutated GH-1 variants (5'IVS-3 + 2-bp T->C; 5'IVS-3 + 6 bp T->C; ISEm1: IVS-3 + 28 G->A) known to cause clinical IGHD II of varying severity. Cells were stimulated with 1 and 10 mum dexamethasone (DEX) for 24 h, after which the relative amounts of GH-1 splice variants were determined by semiquantitative and quantitative (TaqMan) RT-PCR. In the absence of DEX, only around 1% wt-GH-1 transcripts were the 17.5-kDa isoform, whereas the three mutant GH-1 variants produced 29, 39, and 78% of the 17.5-kDa isoform. DEX stimulated total GH-1 gene transcription from all constructs. Notably, however, DEX increased the amount of 17.5-kDa GH isoform relative to the 22- and 20-kDa isoforms produced from the mutated GH-1 variants, but not from wt-GH-1. This DEX-induced enhancement of 17.5-kDa GH isoform production, up to 100% in the most severe case, was completely blocked by the addition of RU486. In other studies, we measured cell proliferation rates, annexin V staining, and DNA fragmentation in cells transfected with the same GH-1 constructs. The results showed that that the 5'IVS-3 + 2-bp GH-1 gene mutation had a more severe impact on those measures than the splice site mutations within 5'IVS-3 + 6 bp or ISE +28, in line with the clinical severity observed with these mutations. Our findings that the proportion of 17.5-kDa produced from mutant GH-1 alleles increases with increased drive for gene expression may help to explain the variable onset progression, and severity observed in IGHD II.

Strategies to prevent neuronal damage in paediatric bacterial meningitis

PURPOSE OF REVIEW: The mortality of bacterial meningitis can reach 30%, and up to 50% of survivors suffer from persisting neurological deficits as a consequence of the disease. The incidence of neurological sequelae of bacterial meningitis has not improved over the last decade. Adjunctive therapeutic options are limited, and ongoing research into the pathophysiology of brain damage in bacterial meningitis aims at providing the scientific basis for future development of more efficient adjunctive options. RECENT FINDINGS: In a population with good access to health care, dexamethasone given before or at the time of initiation of antibiotic therapy acts beneficially in paediatric pneumococcal meningitis, but not in meningococcal meningitis. In experimental animal models, brain-derived neurotrophic factor protected against brain injury and improved hearing while melatonin, which has antioxidant properties among other effects, reduced neuronal death. Transgene technology can be used to provide new insights into the pathophysiology of the disease and to identify potential therapeutic targets. SUMMARY: Although dexamethasone improves outcome of bacterial meningitis under defined circumstances, the morbidity of bacterial meningitis still remains unacceptably high. Experimental models may help to identify new therapeutic strategies to further improve the neurological outcome in young children suffering from bacterial meningitis.

Effects of colostrum feeding and glucocorticoid administration on insulin-dependent glucose metabolism in neonatal calves

Colostrum feeding and glucocorticoid administration affect glucose metabolism and insulin release in calves. We have tested the hypothesis that dexamethasone as well as colostrum feeding influence insulin-dependent glucose metabolism in neonatal calves using the euglycemic-hyperinsulinemic clamp technique. Newborn calves were fed either colostrum or a milk-based formula (n=14 per group) and in each feeding group, half of the calves were treated with dexamethasone (30 microg/[kg body weight per day]). Preprandial blood samples were taken on days 1, 2, and 4. On day 5, insulin was infused for 3h and plasma glucose concentrations were kept at 5 mmol/L+/-10%. Clamps were combined with [(13)C]-bicarbonate and [6,6-(2)H]-glucose infusions for 5.5h (i.e., from -150 to 180 min, relative to insulin infusion) to determine glucose turnover, glucose appearance rate (Ra), endogenous glucose production (eGP), and gluconeogenesis before and at the end of the clamp. After the clamp liver biopsies were taken to measure mRNA levels of phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate carboxylase (PC). Dexamethasone increased plasma glucose, insulin, and glucagon concentrations in the pre-clamp period thus necessitating a reduction in the rate of glucose infusion to maintain euglycemia during the clamp. Glucose turnover and Ra increased during the clamp and were lower at the end of the clamp in dexamethasone-treated calves. Dexamethasone treatment did not affect basal gluconeogenesis or eGP. At the end of the clamp, dexamethasone reduced eGP and PC mRNA levels, whereas mitochondrial PEPCK mRNA levels increased. In conclusion, insulin increased glucose turnover and dexamethasone impaired insulin-dependent glucose metabolism, and this was independent of different feeding.

Tumor necrosis factor-alpha: alternative role as an inhibitor of osteoclast formation in vitro

TNFalpha is known to stimulate the development and activity of osteoclasts and of bone resorption. The cytokine was found to mediate bone loss in conjunction with inflammatory diseases such as rheumatoid arthritis or chronic aseptic inflammation induced by wear particles from implants and was suggested to be a prerequisite for the loss of bone mass under estrogen deficiency. In the present study, the regulation of osteoclastogenesis by TNFalpha was investigated in co-cultures of osteoblasts and bone marrow or spleen cells and in cultures of bone marrow and spleen cells grown with CSF-1 and RANKL. Low concentrations of TNFalpha (1 ng/ml) caused a >90% decrease in the number of osteoclasts in co-cultures, but did not affect the development of osteoclasts from bone marrow cells. In cultures with p55TNFR(-/-) osteoblasts and wt BMC, the inhibitory effect was abrogated and TNFalpha induced an increase in the number of osteoclasts in a dose-dependent manner. Osteoblasts were found to release the inhibitory factor(s) into the culture supernatant after simultaneous treatment with 1,25(OH)(2)D(3) and TNFalpha, this activity, but not its release, being resistant to treatment with anti-TNFalpha antibodies. Dexamethasone blocked the secretion of the TNFalpha-dependent inhibitor by osteoblasts, while stimulating the development of osteoclasts. The data suggest that the effects of TNFalpha on the differentiation of osteoclast lineage cells and on bone metabolism may be more complex than hitherto assumed and that these effects may play a role in vivo during therapies for inflammatory diseases.

Mandibular reconstruction using a combination graft of rhBMP-2 with bone marrow cells expanded in vitro

BACKGROUND: The aim of this study was to evaluate the efficacy of a combination graft, using recombinant human bone morphogenetic protein-2 (rhBMP-2) and culture-expanded cells derived from bone marrow, for bone regeneration in a nonhuman primate mandible. METHODS: Five Japanese monkeys were used. Three milliliters of bone marrow was obtained from the tibia and plated into culture flasks. Adherent cells were cultured until near confluence; then, the proliferated cells were transferred to a three-dimensional culture system using collagen beads as the cell carrier. The medium was supplemented with ascorbic acid, beta-glycerophosphate, and dexamethasone to promote osteoblastic differentiation. After further proliferation on beads, the cells were mixed with a collagen sponge that was impregnated with rhBMP-2 and grafted into surgically created segmental bone defects of the mandibles. Three animals received this treatment, and either culture-expanded cells alone or collagen beads without cells were implanted into the remaining two monkeys as controls. The animals were killed 24 weeks after surgery, and the results were assessed by radiographic and histologic evaluation. RESULTS: The combination graft of culture-expanded bone marrow cells with rhBMP-2 in a collagen sponge regenerated the mandibular bone completely. By contrast, the graft of culture-expanded cells alone resulted in only a small amount of bone formation, and the implantation of collagen beads alone led to no bone formation. CONCLUSION: The combination graft of rhBMP-2 and culture-expanded cells, which requires only a small amount of bone marrow, is a reliable method for the reconstruction of segmental bone defects of the mandible.

Empty sella syndrome, hyperadrenocorticism and megaoesophagus in a dachshund

A five-year-old, entire, male dachshund was presented with a five day history of hypersalivation and regurgitation as well as polyuria and polydipsia for several months. Chest radiographs demonstrated megaoesophagus and aspiration pneumonia. Furthermore, hyperadrenocorticism was demonstrated by means of elevations in levels of serum alkaline phosphatase and cholesterol, decreased urinary specific gravity, increased response to adrenocorticotropic hormone stimulation, insufficient suppression of the post-dexamethasone plasma cortisol levels, an increased endogenous adrenocorticotropic hormone concentration and bilaterally enlarged adrenal glands on abdominal ultrasound. The dog became severely dyspnoeic and was euthanased after magnetic resonance imaging was performed. The magnetic resonance imaging and necropsy revealed the sellar region mainly filled with fluid, with only small tissue remnants, a condition defined as empty sella syndrome in human medicine. To the author's knowledge, this is the first dog described with empty sella syndrome and only the second dog described with hyperadrenocorticism secondary to ectopic adrenocorticotropic hormone production. However, the association between empty sella syndrome and hyperadrenocorticism may be no more than incidental.

Multilineage differentiation potential of equine blood-derived fibroblast-like cells

Tissue engineering (TE) has emerged as a promising new therapy for the treatment of damaged tissues and organs. Adult stem cells are considered as an attractive candidate cell type for cell-based TE. Mesenchymal stem cells (MSC) have been isolated from a variety of tissues and tested for differentiation into different cell lineages. While clinical trials still await the use of human MSC, horse tendon injuries are already being treated with autologous bone marrow-derived MSC. Given that the bone marrow is not an optimal source for MSC due to the painful and risk-containing sampling procedure, isolation of stem cells from peripheral blood would bring an attractive alternative. Adherent fibroblast-like cells have been previously isolated from equine peripheral blood. However, their responses to the differentiation conditions, established for human bone marrow MSC, were insufficient to fully confirm their multilineage potential. In this study, differentiation conditions were optimized to better evaluate the multilineage capacities of equine peripheral blood-derived fibroblast-like cells (ePB-FLC) into adipogenic, osteogenic, and chondrogenic pathways. Adipogenic differentiation using rabbit serum resulted in a high number of large-size lipid droplets three days upon induction. Cells' expression of alkaline phosphatase and calcium deposition upon osteogenic induction confirmed their osteogenic differentiation capacities. Moreover, an increase of dexamethasone concentration resulted in faster osteogenic differentiation and matrix mineralization. Finally, induction of chondrogenesis in pellet cultures resulted in an increase in cartilage-specific gene expression, namely collagen II and aggrecan, followed by protein deposition after a longer induction period. This study therefore demonstrates that ePB-FLC have the potential to differentiate into adipogenic, osteogenic, and chondrogenic mesenchymal lineages. The presence of cells with confirmed multilineage capacities in peripheral blood has important clinical implications for cell-based TE therapies in horses.