Combination of the PI3K inhibitor Idelalisib with the conventional cytostatics cytarabine and dexamethasone leads to changes in pathway activation that induce anti-proliferative effects in B lymphoblastic leukaemia cell lines.

BACKGROUND: The introduction of combined conventional cytostatics and pathway-specific inhibitors has opened new treatment options for several cancer types including hematologic neoplasia such as leukaemias. As the detailed understanding of the combination-induced molecular effects is often lacking, the identification of combination-induced molecular mechanisms bears significant value for the further development of interventional approaches. METHODS: Combined application of conventional cytostatic agents (cytarabine and dexamethasone) with the PI3K-inhibitor Idelalisib was analysed on cell-biologic parameters in two acute pro-B lymphoblastic leukaemia (B-ALL) cell lines. In particular, for comparative characterisation of the molecular signatures induced by the combined and mono application, whole transcriptome sequencing was performed. Emphasis was placed on pathways and genes exclusively regulated by drug combinations. RESULTS: Idelalisib + cytostatics combinations changed pathway activation for, e.g., "Retinoblastoma in cancer", "TGF-b signalling", "Cell cycle" and "DNA-damage response" to a greater extent than the two cytostatics alone. Analyses of the top-20 regulated genes revealed that both combinations induce characteristic gene expression changes. CONCLUSION: A specific set of genes was exclusively deregulated by the drug combinations, matching the combination-specific anti-proliferative cell-biologic effects. The addition of Idelalisib suggests minor synergistic effects which are rather to be classified as additive.

Supporting individuals with intellectual and developmental disability during the first 100 days of the COVID-19 outbreak in the USA.

BACKGROUND: It is unknown how the novel Coronavirus SARS-CoV-2, the cause of the current acute respiratory illness COVID-19 pandemic that has infected millions of people, affects people with intellectual and developmental disability (IDD). The aim of this study is to describe how individuals with IDD have been affected in the first 100 days of the COVID-19 pandemic. METHODS: Shortly after the first COVID-19 case was reported in the USA, our organisation, which provides continuous support for over 11 000 individuals with IDD, assembled an outbreak committee composed of senior leaders from across the health care organisation. The committee led the development and deployment of a comprehensive COVID-19 prevention and suppression strategy, utilising current evidence-based practice, while surveilling the global and local situation daily. We implemented enhanced infection control procedures across 2400 homes, which were communicated to our employees using multi-faceted channels including an electronic resource library, mobile and web applications, paper postings in locations, live webinars and direct mail. Using custom-built software applications enabling us to track patient, client and employee cases and exposures, we leveraged current public health recommendations to identify cases and to suppress transmission, which included the use of personal protective equipment. A COVID-19 case was defined as a positive nucleic acid test for SARS-CoV-2 RNA. RESULTS: In the 100-day period between 20 January 2020 and 30 April 2020, we provided continuous support for 11 540 individuals with IDD. Sixty-four per cent of the individuals were in residential, community settings, and 36% were in intermediate care facilities. The average age of the cohort was 46 ± 12 years, and 60% were male. One hundred twenty-two individuals with IDD were placed in quarantine for exhibiting symptoms and signs of acute infection such as fever or cough. Sixty-six individuals tested positive for SARS-CoV-2, and their average age was 50. The positive individuals were located in 30 different homes (1.3% of total) across 14 states. Fifteen homes have had single cases, and 15 have had more than one case. Fifteen COVID-19-positive individuals were hospitalised. As of 30 April, seven of the individuals hospitalised have been discharged back to home and are recovering. Five remain hospitalised, with three improving and two remaining in intensive care and on mechanical ventilation. There have been three deaths. We found that among COVID-19-positive individuals with IDD, a higher number of chronic medical conditions and male sex were characteristics associated with a greater likelihood of hospitalisation. CONCLUSIONS: In the first 100 days of the COVID-19 outbreak in the USA, we observed that people with IDD living in congregate care settings can benefit from a coordinated approach to infection control, case identification and cohorting, as evidenced by the low relative case rate reported. Male individuals with higher numbers of chronic medical conditions were more likely to be hospitalised, while most younger, less chronically ill individuals recovered spontaneously at home.

Contraction parameters, myosin composition and metabolic enzymes of the skeletal muscles of the etruscan shrew Suncus etruscus and of the common European white-toothed shrew Crocidura russula (Insectivora: soricidae).

In the Etruscan shrew, the isometric twitch contraction times of extensor digitorum longus (EDL) and soleus muscles are shorter than in any other mammal, allowing these muscles to contract at outstandingly high contraction frequencies. This species has the highest mass-specific metabolic rate of all mammals and requires fast skeletal muscles not only for locomotion but also for effective heat production and for an extremely high ventilation rate. No differences could be detected in the fibre type pattern, the myosin heavy and light chain composition, or in the activity of the metabolic enzymes lactate dehydrogenase and citrate synthase of the two limb muscles, the EDL and the soleus, which in larger mammalian species exhibit distinct differences in contractile proteins and metabolic enzymes. All properties determined in EDL and soleus muscles of Suncus etruscus, as well as in the larger Crocidura russula, are typical for fast-oxidative fibres, and the same holds for several other skeletal muscles including the diaphragm muscle of S. etruscus. Nevertheless, the EDL and soleus muscles showed different mechanical properties in the two shrew species. Relaxation times and, in C. russula, time to peak force are shorter in the EDL than in the soleus muscle. This is in accordance with the time course of the Ca(2+) transients in these muscles. Such a result could be due to different parvalbumin concentrations, to a different volume fraction of the sarcoplasmic reticulum in the two muscles or to different Ca(2+)-ATPase activities. Alternatively, the lower content of cytosolic creatine kinase (CK) in the soleus compared with the EDL muscle could indicate that the observed difference in contraction times between these shrew muscles is due to the CK-controlled activity of their sarcoplasmic reticulum Ca(2+)-ATPase.

Carbonic anhydrase in the gills of seawater- and freshwater-acclimated flounders Platichthys flesus: purification, characterization, and immunohistochemical localization.

Flounders Platichthys flesus were investigated with respect to isolation, purification, and cellular localization of carbonic anhydrase (CA) in the respiratory system. CA was purified from gills and erythrocytes and was shown to exclusively represent a soluble enzyme with an apparent molecular weight of 30 kD. Inhibition constants (KI) towards acetazolamide (ACTZ) were 8.4.10(-9) M for erythrocyte CA and 7.6.10(-9) M for gill CA, indicating a high sensitivity to sulfonamides, as exhibited by human CA II. Specific CA activity did not differ significantly in seawater- and freshwater-acclimated fish. Antibodies were raised against purified gill and erythrocyte CA. Both antisera crossreacted and were used to localize CA in the gills of seawater and freshwater flounders at the light microscopic level. Independent of the salinity, a positive reaction of variable intensity was found in the following cell types: pavement cells (PVCs), forming the gill epithelial surface layer; mucous cells (MCs); pillar cells (PCs), bordering the vascular channels of the secondary lamellae; and chloride cells (CCs), mitochondria-rich cells located in the primary epithelium, the interlamellar regions, and at the bases of the secondary lamellae.(J Histochem Cytochem 47:43-50, 1999)

Localization of carbonic anhydrase IV in rat and human heart muscle.

We investigated carbonic anhydrase IV (CA IV) in rat and human heart with immunohistochemical methods by both light and electron microscopy. In cryosections that were incubated with anti-CA IV/FITC, the capillaries showed a strong reaction for CA IV. In paraffin and semithin sections treated with anti-CA IV/ABC (avidin-biotin-peroxidase complex) blood vessels, capillaries, and sarcolemma (SL) were positively stained. By staining ultrathin sections with anti-CA IV/immunogold, CA IV could also be demonstrated at the latter two locations, including the specialized sarcolemmal structures intercalated discs, and T-tubules. In addition, by this method CA IV was seen to be associated with the sarcoplasmic reticulum (SR). The absence of immunostaining in SR and/or SL with some techniques probably indicates a problem of accessibility of the antigenic sites. In line with the immunohistochemical results, CA IV mRNA expression was visualized in both endothelial and muscle cells by in situ hybridization histochemistry.

Rates of rewarming, heart and respiratory rates and their significance for oxygen transport during arousal from torpor in the smallest mammal, the Etruscan shrew Suncus etruscus.

We investigated the process of rewarming from torpor with respect to respiratory and circulatory oxygen transport properties in the smallest mammal, the Etruscan shrew Suncus etruscus. In seven adult Etruscan shrews with a mean body mass of 2.4g, torpor was induced by deprivation of food and a cold environment. During arousal from torpor at an ambient temperature of 22 degrees C, the shrews actively rewarmed from the lowest mean (+/- S.D.) body temperature (Tb) of 12.1 +/- 1.2 degrees C to 20 degrees C at a rate of 0.43 +/- 0.14 degree C min-1, from 20 to 24 degrees C at a rate of 0.8 degree C min-1, and from 24 to 36 degrees C at a rate of 1.1 +/- 0.1 degrees C min-1. The mean rate from 12 degrees C to normothermia amounted to 0.83 degree C min-1, which is among the highest values recorded in mammals. During rewarming, the heart rate increased exponentially (Q10 = 2.2) from 100 to 800-1200 min-1, whereas the respiratory rate increased linearly from 50 to 600-800 min-1. These rates are higher than the heart and respiratory rates reported for other small mammals at the same Tb. The fraction of brown adipose tissue (BAT) was 9.2 +/- 1.6% of body mass, which is higher than in any other mammal. Up to a body temperature of approximately 17 degrees C, the heat for rewarming was mainly produced in the BAT; above this value, considerable activity of the skeletal muscles enhanced thermogenesis. Estimation of the mixed venous oxygen partial pressure showed that, at the tissue level, the rewarming process corresponds to heavy work conditions. The ventilatory system is adapted such that during rewarming, in addition to the appropriate oxygen transport capacity, there is also a capacity for hyperventilation.

Heart and respiratory rates and their significance for convective oxygen transport rates in the smallest mammal, the Etruscan shrew Suncus etruscus.

Heart and respiratory rates of the smallest mammal (mean adult body mass 2g), the Etruscan shrew Suncus etruscus, were determined at rest and under stress conditions. Heart rate was obtained from electrocardiograms (ECGs), recorded via foot electrodes. The mean +/- S.D. heart rate of resting animals (ambient temperature 22 degrees C) was 835 +/- 107 min-1, the mean maximal rate amounted to 1093 +/- 235 min-1. The highest single value recorded was 1511 min-1, which is the highest heart rate reported so far for an endotherm. The respiratory rate was also obtained from ECG recordings, which showed the electrical activity of the breathing muscles during inhalation, and additionally by recording the movements of the thoracic wall with a laser autofocus system. The mean resting respiratory rate was 661 +/- 93 min-1, the mean maximal rate was 758 +/- 109 min-1 and the highest single value recorded was 894 min-1. At 22 degrees C, the specific oxygen consumption rate is 67 times higher in resting S. etruscus than in resting humans. Under these conditions, the respiratory rate of the shrew is 47 times higher but the heart rate only 12 times higher than in man. Therefore, to achieve an adequate circulatory oxygen transport rate, the product of relative stroke volume and arterio-venous O2 difference has to be 5.6 times higher in the shrew than in man, whereas for an appropriate ventilatory oxygen transport rate the product of relative tidal volume and oxygen extraction has to be only 1.4 times higher in this small insectivore than in man. The maximal possible oxygen transport rates of the ventilatory and the circulatory system have been estimated and compared with the diffusional transport capacity of the lung. These rates amount to approximately 1000 ml O2 kg-1 min-1. According to our results and data in the literature, an aerobic scope of 7-10 seems to be realistic for the Etruscan shrew.

Membrane-associated carbonic anhydrase IV in skeletal muscle: subcellular localization.

Carbonic anhydrase IV (CA IV) was examined by light microscopy and electron microscopy in rat soleus muscle. Semithin sections of aldehyde-fixed Epon-embedded muscle were stained with rabbit anti-rat lung CA IV and the avidin-biotin-peroxidase complex. With this technique, capillaries and sarcolemma showed positive CA IV staining. For electron microscopy, rat soleus specimens were aldehyde-fixed, with or without subsequent osmication, and embedded in Epon. Ultrathin sections were immunostained with anti-rat lung CA IV/immunogold. Omitting osmium allowed ample antigen-antibody reactions but could not prevent the release of glycosylphosphatidylinositol-anchored CA IV from the membranes, which led to apparent background staining. Postosmication significantly reduced tissue antigenicity but kept the antigen bound to the membranes and thus allowed a very precise localization of CA IV. By electron microscopy, membrane-bound CA IV is found to be associated with capillary endothelium, sarcolemma, and sarcoplasmic reticulum (SR). Conceivably, the presence of SR staining in ultrathin sections and its absence in semithin sections reflect a problem of accessibility of the antigenic sites.

Immunohistochemical localization of carbonic anhydrase IV in capillaries of rat and human skeletal muscle.

We used polyclonal antisera raised in rabbits against membrane-bound rat lung and human lung carbonic anhydrase (CA) IV in immunofluorescence studies to stain cryosections of rat soleus and extensor digitorum longus (EDL) and several human skeletal muscles. There was strong specific staining of capillaries in all muscles investigated. Several techniques were applied to verify this result. (a) Serial sections were either incubated with anti-CA IV/FITC or processed for endothelial ATPase reaction. There was precise co-localization of antibody marked structures and ATPase stained capillaries. (b) Human muscle sections were double stained with anti-CA IV/TRITC and anti-von Willebrand factor (vWF)/FITC. vWF, a capillary marker, and CA IV were localized at identical sites. (c) The CAIV was released from capillaries by treatment with phosphatidylinositol specific phospholipase C, suggesting that the enzyme is anchored to the endothelial cell membrane via a phosphatidylinositolglycan anchor. (d) A rat hindlimb was perfused with diluted antiserum. Cryosections of perfused soleus and EDL processed for anti-rabbit IgG/FITC staining showed clear fluorescence associated with capillaries, indicating that the antigen was accessible from the capillary lumen. (e) Immune complexes formed during antiserum perfusion as described in d were precipitated from muscle homogenates. SDS-PAGE followed by immunoblotting showed that the predominant portion of total muscle CA IV was bound in these complexes and therefore must be located intravascularly.