3D printed, personalized sustained release cortisol for patients with adrenal insufficiency.

The standard of care for patients with Adrenal Insufficiency (AI) is suboptimal. Administration of hydrocortisone three times a day produces plasma cortisol fluctuations associated with negative health outcomes. Furthermore, there is a high inter-individual variability in cortisol need, necessitating a personalized approach. It is hypothesized that a personalized, sustained release formulation would enhance the pharmacotherapy by mimicking the physiological cortisol plasma concentration at a higher level. Therefore, a novel 24 h sustained release 3D printed (3DP) hydrocortisone formulation has been developed (M3DICORT) by coupling hot-melt extrusion with fused deposition modeling. A uniform drug distribution in the 3DP tablets is demonstrated by a content of 101.66 ± 1.60 % with an acceptance value of 4.01. Furthermore, tablets had a stable 24 h dissolution profile where the intra-batch standard deviation was ± 2.8 % and the inter-batch standard deviation was ± 6.8 %. Tablet height and hydrocortisone content were correlated (R2 = 0.996), providing a tool for easy dose personalization. Tablets maintained critical quality attributes, such as dissolution profile (f2 > 60) and content uniformity after process transfer from a single-screw extruder to a twin-screw extruder. Impurities were observed in the final product which should be mitigated before clinical assessment. To our knowledge, M3DICORT is the first 3DP hydrocortisone formulation specifically developed for AI.

Identification of an immune-responsive mesolimbocortical serotonergic system: potential role in regulation of emotional behavior.

Peripheral immune activation can have profound physiological and behavioral effects including induction of fever and sickness behavior. One mechanism through which immune activation or immunomodulation may affect physiology and behavior is via actions on brainstem neuromodulatory systems, such as serotonergic systems. We have found that peripheral immune activation with antigens derived from the nonpathogenic, saprophytic bacterium, Mycobacterium vaccae, activated a specific subset of serotonergic neurons in the interfascicular part of the dorsal raphe nucleus (DRI) of mice, as measured by quantification of c-Fos expression following intratracheal (12 h) or s.c. (6 h) administration of heat-killed, ultrasonically disrupted M. vaccae, or heat-killed, intact M. vaccae, respectively. These effects were apparent after immune activation by M. vaccae or its components but not by ovalbumin, which induces a qualitatively different immune response. The effects of immune activation were associated with increases in serotonin metabolism within the ventromedial prefrontal cortex, consistent with an effect of immune activation on mesolimbocortical serotonergic systems. The effects of M. vaccae administration on serotonergic systems were temporally associated with reductions in immobility in the forced swim test, consistent with the hypothesis that the stimulation of mesolimbocortical serotonergic systems by peripheral immune activation alters stress-related emotional behavior. These findings suggest that the immune-responsive subpopulation of serotonergic neurons in the DRI is likely to play an important role in the neural mechanisms underlying regulation of the physiological and pathophysiological responses to both acute and chronic immune activation, including regulation of mood during health and disease states. Together with previous studies, these findings also raise the possibility that immune stimulation activates a functionally and anatomically distinct subset of serotonergic neurons, different from the subset of serotonergic neurons activated by anxiogenic stimuli or uncontrollable stressors. Consequently, selective activation of specific subsets of serotonergic neurons may have distinct behavioral outcomes.

The human factor H-related protein 4 (FHR-4). A novel short consensus repeat-containing protein is associated with human triglyceride-rich lipoproteins.

A novel apoprotein of an apparent molecular mass of 86 kDa in its unreduced form was identified in human triglyceride-rich lipoproteins. This protein was purified and the amino acid sequence of six proteolytic fragments was found to overlap with that of the factor H-related proteins. In parallel we identified the cDNA encoding a new complement factor H-related protein, termed FHR-4. The sequences of the new apoprotein overlapped with that of the FHR-4 protein. Similar to the previously described factor H-related proteins, FHR-4 contains a hydrophobic signal sequence followed by a stretch of five repetitive elements termed short consensus repeats. Recombinant FHR-4 protein was expressed in the baculovirus system and has an apparent molecular mass of 42 kDa. In addition a 84-kDa dimeric form of the recombinant FHR-4 was detected. Using an immunoaffinity column with antibodies raised against the recombinant FHR-4, we isolated a 86-kDa protein from human plasma. The different molecular mass of the recombinant FHR-4 and the dimeric FHR-4 in plasma is due to different carbohydrate moieties. The 86-kDa plasma protein and the novel apolipoprotein had identical mobility on SDS-polyacrylamide gel electrophoresis analysis and reacted with antisera raised against the reFHR-4 and the purified apoprotein. In conclusion, we have identified a novel factor H-related protein, FHR-4, in human plasma and demonstrate that this protein is present in triglyceride-rich lipoproteins in a dimeric form. This observation provides an intriguing new aspect on possible function(s) of this novel protein and the other factor H-related proteins.

Role of telomeric sequences in murine radiation-induced myeloid leukaemia.

A previous study indicated that a highly inbred CBA/H mouse colony contained four genotypic variants for telomere-like repeat (TLR) sequence arrays and that one variant subpopulation that constituted 20% of the colony contributed the vast majority (> 90%) of radiation-induced acute myeloid leukaemias (AMLs). Through screening of a satellite CBA/H colony and rescreening of the original colony, we show that, whereas germline telomere sequence polymorphism is frequent in CBA/H mice, there is no genetic link between a specific TLR locus variant and susceptibility to AML. Studies on telomere-hybridising fragments between 200 bp and 150 kb revealed that the germline telomere mutation frequency was highest for restriction fragments > 50 kb. The hypervariability of these high-molecular-weight fragments resulted in each CBA/H mouse from the highly inbred colony having a different genotype. Although it was not possible to ascribe a specific somatic telomere mutation to AML development, telomere rearrangements were common in induced AMLs. Some terminal telomere-hybridising restriction fragments were shortened in AML samples in comparison with normal tissue, but, insofar as the reduction in size was relatively small, it seems unlikely that telomere erosion is a major contributor to the molecular pathology of murine radiation-induced AML.

Human 55-kDa receptor for tumor necrosis factor coupled to signal transduction cascades.

The numerous biological activities of tumor necrosis factor (TNF) appear mediated by two types of receptors of 55 kDa (TR55) and 75 kDa (TR75) molecular mass. To test TR55 for its individual role in signaling across the membrane, a cDNA coding for the human TR55 was stably expressed in murine 70Z/3 pre-B cells, which lack binding sites for, and proved nonresponsive to human TNF. The transfected TR55 showed high affinity ligand binding and active internalization. It is demonstrated that the TNF signaling cascade, i.e. stimulation of protein kinase C, sphingomyelinase, and phospholipase A2, production of the second messengers diacylglycerol and ceramide, can occur completely through exclusive binding of TNF to TR55. The p55 TNF-binding site functions as an autonomous TNF receptor that mediates key signal transduction pathways, which may control the majority of TNF actions.

Results of routine determination of clinically significant hemoglobin derivatives by multicomponent analysis.

Our recently developed multiwavelength method for multi-component analysis of hemoglobin (Hb) derivatives (Clin Chem 1984;30:373-379) was adapted for routine use in the clinical chemical laboratory. The method was applied in 4066 determinations on blood specimens from patients awaiting major surgery (n = 3863) or visiting the outpatient department for pulmonary disease (n = 203). Mean total hemoglobin concentration was 141 (SD 14) g/L. The proportion of HbCO was slightly to moderately increased (1.5-10.0%) in 36.5% of all patients; in a few cases it was as high as 15%. Mean methemoglobin was 0.4% (SD 0.2%) in the surgical patients, but 1.5% (SD 0.8%) in the patients with pulmonary disease. In some patients of the latter group the proportion of methemoglobin amounted to 5%. Sulfhemoglobin was found less than 0.4% in all specimens. Interference by paraproteins and by increased concentrations of bilirubin and lipids in plasma was easily detected by means of the performance checks provided by the spectrophotometer (an HP 8451 UV/Vis). The method is equally suitable for measuring blood samples containing fetal hemoglobin.

Simultaneous spectrophotometric calibration of wavelength and absorbance in an interlaboratory survey using holmium oxide (Ho2O3) in perchloric acid as reference, compared with p-nitrophenol and cobaltous sulphate solutions (1978-1984).

The wavelength accuracy of ten different types of spectrophotometer was tested with holmium perchlorate solutions. It was found to be good, with mean deviations from the literature values of maximally 0.3 nm. Standard deviations over the entire spectral range were within 0.75 nm. The absorbance accuracy for different types of instruments was generally within 5%, except in the 287 nm range where higher deviations were found. The sharpness of the holmium peaks, in combination with band width and sensitivity of the instruments, troubled the majority of the participants. 150 spectrophotometers were involved in the surveys. Linearity of the spectrophotometers was tested with p-nitrophenol and cobaltous sulphate and found to be satisfactory.

Multicomponent analysis of hemoglobin derivatives with reversed-optics spectrophotometer.

We describe a method for the simultaneous determination of the five clinically relevant hemoglobin derivatives (Hb, HbO2, HbCO, Hi, SHb) in a blood sample by means of a reversed-optics spectrophotometer (Hewlett-Packard HP8450 A UV/Vis). A built-in computer program is used for multicomponent analysis in an overdetermined system, i.e., a system in which the number of independent equations used exceeds the number of unknowns to be determined. First, the spectra of the five hemoglobin derivatives are measured in a series of different human blood samples. Thereafter, the multicomponent method for the simultaneous determination of the five hemoglobin derivatives is tested by comparison with conventional methods for the separate determination of oxygen saturation, HbCO, Hi, and SHb fractions. The multicomponent (multiwavelength) method is sufficiently reliable, accurate, and easy to justify its use in physiological chemical research as well as its routine application in the clinical chemical laboratory.

Interlaboratory investigation on the CEA assay (Roche) with column filtration, dialysis and ultrafiltration techniques.

An interlaboratory study on the reproducibility of the CEA (Roche) RIA Test was carried out. Four different plasma pools of approximately 2, 3, 6, and 12 micrograms/l CEA were tested over a period of 4 weeks with 4 different lots of reagents in order to determine the interassay variances. At the same time we compared the lately introduced column technique with the dialysis and ultrafiltration method. Best results were obtained with the column technique which also showed best reproducibility. Only 1.4% of samples showed deviations greater than 5% between the mean of CEA duplicates and single CEA values, and these were omitted from the evaluation. On the other hand about 15% of the corresponding dialysis results showed deviations greater than 5% and were excluded from the evaluation. The methods compared showed a good correlation with a coefficient of 0.96, but the average values for the CEA determination, using the columns technique were lower than those obtained from dialysis. Interassay variances were greater for the dialysis procedures, i.e. 1.88 +/- 0.81, 3.25 +/- 0.83, 5.81 +/- 1.09, and 11-91 +/- 1.23 compared with 1.77 +/- 0.54, 2.63 +/- 0.68, 4.89 +/- 0.79, and 11.16 +/- 1.23 for the column technique. There were no systematic changes of the CEA values over the period of 4 weeks, thus giving optimal conditions for a follow up of patients.

A multi-wavelength spectrophotometric method for the simultaneous determination of five haemoglobin derivatives.

A method is described by which the concentration of deoxyhaemoglobin, oxyhaemoglobin, carboxyhaemoglobin, haemoglobin and sulfhaemoglobin in a human blood sample is determined by passing the haemolysate without air contact through a coarse filter and subsequently measuring the absorbance at lambda = 500, 569, 577, 620 and 760 nm. The ensuing set of equations is solved by matrix calculation with the aid of a simple computer program. The method has been tested by comparing it with conventional methods for the determination of the various haemoglobin derivatives separately.

Ionic composition of arterial and mixed venous plasma in the unanesthetized dog.

The use of permanent catheters in the aorta and pulmonary artery permitted the establishment of normal values for hemoglobin concentration in blood and -or pH, PCO2, osmolality, and protein and electrolyte concentrations in the plasma of arterial and venous samples from unanesthetized, undisturbed dogs, and the comparison of the ionic composition of simultaneously taken arterial and venous samples. Arterial samples yielded the following mean values: CHb, 143 g liter-1; pHP, 7.427; PCO2, 32.5 mmHg; CPosmol, 295 mmol kg-1; CPpr, 73.1 g liter-1, CPNa+, 148.0; CPK+, 3.9; CPCa2+, 2.38; CPMg2+, 0.85; CPCl-, 116.0; CPHCO3-, 21.1; CPlact-, 1.4; CPphosph, 1.21; net cation equivalency, 16.4; and anion gap, 1.03 mmol liter-1 in eight male mongrel dogs with seven or eight samplings from each dog. The anion gap in arterial and venous plasma was small, indicating that the contribution of sulfate and organic acids to the ionic composition of dog plasma is quantitatively unimportant. In simultaneously taken arterial and venous samples the following significant arteriovenous differences were found: HP, +0.038; PCO2, -5.6 mmHg; CPosmol, -1.8 mmol kg-1; protein, -0.8 g liter-1; CPNa+, -1.0; CPK+, -0.1; CPCl-, +1.3; and CPHCO3-, -1.7 mmol liter-1. These differences are explained on the basis of the changes that occur in blood upon the addition of CO2 and the ensuing chloride and water shifts.

Carboxyhaemoglobin. Spectrophotometric determination tested and calibrated using a new reference method for measuring carbon monoxide in blood.

The spectrophotometric determination of HbCO at lambda = 562 and 540 nm in the system HbCO/HbO2 was reinvestigated using a new reference method for measuring CO in blood. This reference method is based on the conversion of CO from HbCO into CO2 which is determined by titration. Plotting the absorbance ratio A562/A540 against the titrimetrically determined HbCO fractions of 46 blood samples demonstrated a linear relationship up to 90% HbCO and yielded more accurate values for the constants in the equation for calculating the HbCO fraction from A562/A540. The standard deviation of the differences between the spectrophotometric and the titrimetric method was 1.2% HbCO. It is shown that the influence of other haemoglobin derivatives and other possible sources of error is either negligible or can be prevented by simple precautions.