Cell cycle regulation of the human Six1 homeoprotein is mediated by APC(Cdh1).

The Six1 homeoprotein is an important mediator of normal development, where it is critical for the proliferation of precursor cell populations that ultimately constitute the muscle, kidney and inner ear, among other organs. Interestingly, its overexpression has been observed in numerous cancers, where it contributes to the proliferative and metastatic ability of the cancer cells. Here we show that Six1 not only regulates the cell cycle, but is itself regulated throughout the cell cycle via ubiquitin-mediated proteolysis. The protein is present from the G(1)/S boundary until mitosis, when it is degraded via the anaphase-promoting complex (APC) with its activating subunit Cdh1. However, unlike most identified APC(Cdh1) targets, Six1 does not contain functional destruction or KEN box motifs that are necessary for its degradation. Instead, the Six1 protein contains multiple, as yet undefined, sequences within its N- and C-termini responsible for its degradation, including an N-terminal region that binds to Cdh1. Cell cycle regulation of Six1 occurs both transcriptionally and post-translationally via phosphorylation; therefore, this study demonstrates a third and novel mechanism of cell cycle-specific regulation of Six1, underscoring the importance of confining its activity to a defined cell cycle window from the G(1)/S boundary to early mitosis.

Interleukin-5 (IL-5) and IL-6 define two molecularly distinct pathways of B-cell differentiation.

Interleukin-5 (IL-5) and IL-6 have both been reported to act as B-cell differentiation factors by stimulating activated B cells to secrete antibody. However, it has not been possible to directly compare the effects of these two lymphokines because of the lack of a suitable B-cell line capable of responding to both. We have identified a clonal, inducible B-cell lymphoma, CH12, that has this property. Both IL-5 and IL-6 can independently stimulate increases in steady-state levels of immunoglobulin and J-chain mRNA and proteins, and they both induce the differentiation of CH12 into high-rate antibody-secreting cells. Nevertheless, there are significant differences in the activities of these two lymphokines. First, while IL-6 acts only as a differentiation factor, IL-5 also augments the proliferation of CH12 cells. Second, the differentiation stimulated by IL-5 but not by IL-6 is partially inhibited by IL-4. Inhibition of IL-5-induced differentiation was not at the level of IL-5 receptor expression, since IL-4 did not inhibit IL-5-induced proliferation. Third, IL-5 but not IL-6 stimulated increased mouse mammary tumor proviral gene expression in CH12 cells. These results demonstrate that while both IL-5 and IL-6 may act as differentiation factors for B cells, they induce differentiation by using at least partially distinct molecular pathways. Our results also establish that B cells characteristic of a single stage of development can independently respond to IL-4, IL-5, and IL-6.

Comparison of 2 Doses for ACTH Stimulation Testing in Dogs Suspected of or Treated for Hyperadrenocorticism.

BACKGROUND: Lowering the cosyntropin dose needed for ACTH stimulation would make the test more economical. OBJECTIVES: To compare the cortisol response to 1 and 5 µg/kg cosyntropin IV in dogs being screened for hyperadrenocorticism (HAC) and in dogs receiving trilostane or mitotane for pituitary-dependent HAC. ANIMALS: Healthy dogs (n = 10); client-owned dogs suspected of having HAC (n = 39) or being treated for pituitary-dependent HAC with mitotane (n = 12) or trilostane (n = 15). PROCEDURES: In this prospective study, healthy dogs had consecutive ACTH stimulation tests to ensure 2 tests could be performed in sequence. For the first test, cosyntropin (1 µg/kg IV) was administered; the second test was initiated 4 hours after the start of the first (5 µg/kg cosyntropin IV). Dogs suspected of having HAC or being treated with mitotane were tested as the healthy dogs. Dogs receiving trilostane treatment were tested on consecutive days at the same time post pill using the low dose on day 1. RESULTS: In dogs being treated with mitotane or trilostane, the 2 doses were pharmacodynamically equivalent (90% confidence interval, 85.1-108.2%; P = 0.014). However, in dogs suspected of having HAC, the doses were not pharmacodynamically equivalent (90% confidence interval, 73.2-92.8%; P = 0.37); furthermore, in 23% of the dogs, clinical interpretation of test results was different between the doses. CONCLUSIONS AND CLINICAL RELEVANCE: For dogs suspected of having HAC, 5 µg/kg cosyntropin IV is still recommended for ACTH stimulation testing. For dogs receiving mitotane or trilostane treatment, a dose of 1 µg/kg cosyntropin IV can be used.

Zinc sensing by metal-responsive transcription factor 1 (MTF1) controls metallothionein and ZnT1 expression to buffer the sensitivity of the transcriptome response to zinc.

Only a small number of genes are known direct targets of the zinc-responsive transcription factor MTF1; therefore, the aim of this study was to gain a more complete understanding of the MTF-1 regulated zinc-responsive component of the transcriptome. A targeted siRNA was used to deplete MTF1 expression in the human intestinal cell line Caco-2. We predicted that the response to zinc of direct MTF1 target genes would be abrogated by MTF1 knockdown. Surprisingly, a greater number of genes were regulated by zinc following MFT1 knockdown, and most genes that responded to zinc under both control and MTF1-depleted conditions had an augmented response in the latter condition. Exceptions were the zinc effluxer ZnT1 and a suite of metallothionein genes, suggesting that responses of other genes to zinc are usually buffered by increases in these proteins. We propose that MTF1 heads a hierarchy of zinc sensors, and through controlling the expression of a raft of metallothioneins and other key proteins involved in controlling intracellular zinc levels (e.g. ZnT1) alters zinc buffering capacity and total cellular zinc content. We tested and validated this model by overexpressing metallothionein and observing the predicted curtailment in response of the zinc-repressed SLC30A5 (ZnT5) promoter. The model provides the framework for an integrated understanding of cellular zinc homeostasis. Because MTs can bind metals other than zinc, this framework links with overall cellular metal homeostasis.

Evaluation of thyroid-stimulating hormone, total thyroxine, and free thyroxine concentrations in hyperthyroid cats receiving methimazole treatment.

BACKGROUND: Iatrogenic hypothyroidism (IH) after treatment of hyperthyroidism can impair renal function. No study compared the efficacy of measurement of serum free thyroxine by equilibrium dialysis (fT4ed) or thyroid-stimulating hormone (TSH) concentrations for monitoring cats receiving methimazole. OBJECTIVES: To (1) compare the ability of total T4 and fT4ed concentrations in conjunction with TSH to define thyroid function in hyperthyroid cats receiving methimazole, (2) determine the prevalence of IH in cats receiving methimazole, and (3) examine the relationship between thyroid axis hormones and serum creatinine concentration. ANIMALS: One hundred and twenty-five serum samples from hyperthyroid cats receiving methimazole and total T4 concentrations ≤3.9 µg/dL. METHODS: Total T4, fT4ed, and TSH concentrations were measured to evaluate thyroid status and serum creatinine concentration was measured to assess renal function. A low total T4 or fT4ed concentration in combination with an increased TSH concentration defined IH. RESULTS: Forty-one cats (33%) had increased TSH concentrations. Of cats with total T4 and fT4ed concentrations below the reference range, 68% and 73%, respectively, had TSH concentrations above the reference range. Only 18% of cats with a normal TSH concentration had an increased serum creatinine concentrations as compared to 39% of those with increased TSH concentrations (P < .001). CONCLUSIONS: Free T4ed does not identify more cats with potential IH as compared to total T4. The IH prevalence was approximately 20%. Measurement of TSH may be more helpful in indicating that azotemia, if present, is at least in part related to IH. Investigation is needed to define TSH assay utility in identifying possible subclinical IH.

The effects of astigmatism and working distance on optic nerve head images using a Heidelberg Retina Tomograph scanning laser ophthalmoscope.

PURPOSE: To determine effects of astigmatism and working distance on optic nerve head images in normal patients using the Heidelberg Retina Tomograph. METHODS: The optic disks of 51 normal healthy subjects, aged 19 to 44 years, were imaged through dilated pupils. Subjects with 0.75 DC or less of astigmatism were imaged without correction at a working distance of 15 mm. They were then re-imaged with a cylindrical correction of +3.00 DC at 90 degrees axis (n = 20). Naturally astigmatic subjects with more than 1.00 DC were imaged without correction and then re-imaged once this was neutralized with their appropriate spectacle prescription (n = 15). The effects of working distance were studied using subjects with 0.75 DC or less (n = 16). Two working distances were used, 15 and 25 mm. At each session the means of three topographic images were taken from which standard deviations and parameters were recorded. Parameters analyzed included cup shape measure, rim area, and inferior temporal rim volume. Z-profile full width at half maximum was calculated from one image per subject for each condition. RESULTS: No significant difference was found in the measured parameters of the optic disk for any astigmatic condition or changes in working distance (P >.05), (paired t test). Both the standard deviation of the mean topographic images and the Z-profile half-maximum width of the axial intensity profile were significantly greater with induced astigmatism of +3.00 DC (P values 0.3 and.00, respectively). CONCLUSIONS: Optic disk parameters are not significantly affected by uncorrected astigmatism (up to 2.50 DC) or working distance. The algorithm used by the Heidelberg Retina Tomograph to generate topographic maps is sufficiently robust that astigmatism up to 2.50 DC does not require correction.

The dexamethasone suppression test and depressive signs in dementia.

The non-suppression by dexamethasone of endogenous cortisol production has been held to be a specific and sensitive indicator of biological depression. Non-suppression has, however, been reported in a proportion of patients with severe dementia. In the present study failure of suppression was found in 10 out of 20 demented patients. The non-suppressors scored significantly higher on a scale of signs of depression. Following antidepressant treatment, 3 out of 8 non-suppressors reverted to normal suppression, but this was not associated with clinical improvements. The implications of these findings are discussed.

Assessment of arteriovenous fistulae from pressure and thermal dilution studies: clinical experience in forearm fistulae.

Knowledge of useful fistula flow (UFF), the maximum blood flow available using twin needles within acceptable pressure limits and without recirculation, is essential for the optimal management of patients receiving maintenance hemodialysis or hemofiltration. A technique for the measurement of UFF employing thermal dilution for the detection of recirculation has been developed. Using this technique, 94 studies were carried out in 69 forearm fistulae. UFF exceeded 400 ml/min in 45 fistulae. In these satisfactory fistulae a normal range was defined for basal intrafistula pressures (60 mmHg being the approximate upper limit of normal). Analysis of arterial and venous line pressure recordings with increasing extracorporeal blood flow and knowledge of the presence or absence of recirculation allowed us to define the functional problem in unsatisfactory fistulae. The functional diagnosis was supported in 16 of 24 fistulae by angiography or surgery or both. In all but two of the remainder, satisfactory UFF was obtained by repositioning the patients' needling sites. Fistulae were divided into different clinical groups. Of 35 fistulae which were thought to be clinically acceptable, 7 were found to be unsatisfactory. In 17 fistulae in patients with poor biochemical control, recirculation was detected in 8. Of 11 fistulae reported to produce poor flows on dialysis, 4 had UFF above 400 ml/min. Of 6 fistulae in patients experiencing needling difficulties, 5 had satisfactory UFF. These studies which take only a few minutes and can be carried out immediately preceding a routine dialysis session not only identify unsatisfactory fistulae, but yield valuable diagnostic information in these cases. This has reduced dependence on angiography and has led to more careful selection of patients for surgery.

Dialysis-induced change in erythrocyte volume: effect on change in blood volume calculated from packed cell volume.

Blood volume (BV) change during hemodialysis is often monitored by packed cell volume (PCV). This assumes erythrocyte volume is constant. We tested this by dialyzing 5 patients for 2 hours against high (154 mmol/l), normal (140 mmol/l) and low (126 mmol/l) dialysate sodium concentrations. Erythrocyte water content, calculated from measured blood and plasma water contents, decreased with high and increased with low dialysate sodium concentrations. Erythrocyte volume, calculated from mean corpuscular hemoglobin concentration (MCHC) decreased 3.8% with high concentration dialysate and increased 2.5% when dialysate concentration was low. These changes correlated significantly (r = 0.80, p less than 0.01) with alterations in plasma sodium. Mean corpuscular volume (MCV), measured with a Coulter-S Plus Counter did not alter because of a methodological artefact. BV change can be calculated from PCV when plasma concentrations of osmotically active substances are changed only if allowance is made for altered erythrocyte volume.

Problem identification in apparently well neonates: implications for early discharge.

The frequency, time of identification, and type of problems of newborns in an urban indigent population were prospectively studied during their hospital stay to evaluate feasibility of early hospital discharge. Eight percent (563) of 7,021 term and near-term low-risk infants developed one or more predefined problems. Of those with problems, 42.1% received therapy and/or a higher level of care. Tachypnea, temperature instability, and cyanotic episodes were the most frequently treated problems. Nearly 69% of all problems were detected after the initial examination, and 31% developed problems after 24 hours of age; 5% were transferred to the NICU. Problems occurring after 24 hours of age emphasize the need for follow-up within days after hospital discharge in this population.

The assessment of arteriovenous fistulae created for haemodialysis from pressure and thermal dilution measurements.

A technique is described for the assessment of arteriovenous fistulae created for haemodialysis. This involves the measurement of intrafistula pressures and 'useful fistula flow' (UFF). The latter we define as the maximum blood flow available for twin needle haemodialysis without recirculation and without unacceptable pressures in the arterial ('A') and venous ('V') lines. The test circuit resembles that used for conventional haemodialysis except there is 'A' and 'V' line pressure and temperature monitoring and no dialyser. Intrafistula pressures are first measured at the time of insertion of the fistula needles. 'A' and 'V' line pressures are then recorded as the extracorporeal blood flow rate is increased in increments from zero to 500 ml/min. A check for recirculation is made at each flow rate. A bolus of cold saline injected into the 'V' line causes a momentary decrease in 'A' line temperature when recirculation is present; when there is no recirculation, 'A' line temperature remains constant. The blood flow rate at which recirculation is first detected will be above the useful fistula flow by definition. This technique allows identification of those patients who obtain high blood flows at the expense of recirculation and thus dialyse inefficiently. Combined pressure and thermal dilution measurements yield valuable information in the investigation of failing or problem fistulae.

Fluid balance modelling in patients with kidney failure.

In patients with kidney failure, adequate control of fluid status remains one of the most difficult routine issues to be addressed in the modern style of dialysis. This is primarily due to the lack of quantitative methods for the assessment of fluid status and the reliance on subjective criteria. Fluid is removed from the blood during dialysis treatments using a process called ultrafiltration. The last decade has seen considerable developments in blood volume monitoring (BVM) technology which has enabled responses to ultrafiltration to be continually monitored on an individual basis. This has enabled feedback control of patients' blood volume to be applied with partial success, reducing the number of symptoms. The feedback control algorithms employed have been relatively unsophisticated, using simple proportional control with no attempt to include models of the patient fluid dynamics. This paper describes the development of some prototype fluid kinetic models which may be used in a more advanced control system. Initial results demonstrate the importance of active control processes in the patients' physiological compensatory mechanisms.

Bilateral idiopathic avascular necrosis of the scaphoid: a rare case of Preiser's disease.

Whereas avascular necrosis of the scaphoid after a fracture is well-documented, idiopathic avascular necrosis of the scaphoid (Preiser's disease) is rare. Little is known of the aetiology of the condition and even less about the best course of management. We describe a rare case of bilateral Preiser's disease. Possible aetiological factors and a summary of the current concepts of management are discussed.

Killed Mycobacterium vaccae suspension in children with moderate-to-severe atopic dermatitis: a randomized, double-blind, placebo-controlled trial.

BACKGROUND: The hygiene hypothesis is often proposed to explain the high prevalence of atopy in the western world. Dysregulation of the immune system may result from inadequate exposure to micro-organisms such as mycobacteria. A small trial suggested that a killed extract of Mycobacterium vaccae ameliorates atopic dermatitis (AD). OBJECTIVES: To confirm in a large clinical trial whether killed M. vaccae ameliorates AD in 5-16-year-old children. METHODS: This was a randomized, placebo-controlled, double-blind, multi-centre study of the effect of intradermal injection of killed M. vaccae (0.1 or 1 mg) on patients, aged 5-16, with moderate-to-severe AD. Patients were followed up for 24 weeks. The primary end point was the change in severity of AD at 12 weeks, assessed using the six area, six-sign, atopic dermatitis (SASSAD) score. Secondary end points included changes in disease extent, patient's global assessment and children's dermatology life quality index. RESULTS: There were 166 patients randomized. The mean SASSAD score fell to a similar degree at week 12 in all treatment arms: from 33 to 24, (26%) in the high-dose group, from 30 to 23 (25%) in the low-dose group and from 36 to 27 (24%) in the placebo group (P>0.05). Secondary end points followed the same trend. Adverse events were generally those expected to occur in this population. Injection site reactions occurred in 32 patients at week 4. CONCLUSIONS: M. vaccae was no more effective than the placebo in ameliorating the severity of AD.

Cutaneous IgA-associated vasculitis induced by alcohol.

A patient with elevated levels of serum IgA developed purpuric lesions histologically resembling Henoch-Schönlein purpura brought on by consuming alcohol. Alcohol challenge with 5 units of alcohol reproduced the lesions, with a rapid rise of circulating CD4+ and CD8+ T cells followed by a fall of serum IgA and C3 concentration. The skin lesions and serum abnormalities resolved spontaneously within 6 weeks of the alcohol challenge.