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Background: NGR–hTNF exploits the peptide asparagine–glycine–arginine (NGR) for selectively targeting tumour necrosis factor (TNF) to CD13-overexpressing tumour vessels. Maximum-tolerated dose (MTD) of NGR–hTNF was previously established at 45  μ g m −2 as 1-h infusion, with dose-limiting toxicity being grade 3 infusion-related reactions. We explored further dose escalation by slowing infusion rate (2-h) and using premedication (paracetamol). Methods: Four patients entered each of 12 dose levels ( n =48; 60–325  μ g m −2 ). Pharmacokinetics, soluble TNF receptors (sTNF-R1/sTNF-R2), and volume transfer constant ( K trans ) by dynamic imaging (dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI)) were assessed pre- and post-treatment. Results: Common related toxicity included grade 1/2 chills (58%). Maximum-tolerated dose was not reached. Both C max ( P <0.0001) and area under the plasma concentration–time curve ( P =0.0001) increased proportionally with dose. Post-treatment levels of sTNF-R2 peaked significantly higher than sTNF-R1 ( P <0.0001). Changes in sTNF-Rs, however, did not differ across dose levels, suggesting a plateau effect in shedding kinetics. As best response, 12/41 evaluable patients (29%) had stable disease. By DCE-MRI, 28/37 assessed patients (76%) had reduced post-treatment K trans values ( P <0.0001), which inversely correlated with NGR–hTNF C max ( P =0.03) and baseline K trans values ( P <0.0001). Lower sTNF-R2 levels and greater K trans decreases after first cycle were associated with improved survival. Conclusion: asparagine–glycine–arginine–hTNF can be safely escalated at doses higher than MTD and induces low receptors shedding and early antivascular effects.

Abstract Disclosure: M. Bertossi: None. C. Lowe: None. Q.L. Van Meter: Consulting Fee; Self; Eton Pharmaceuticals. Speaker; Self; Eton Pharmaceuticals. Introduction: Congenital adrenal hyperplasia (CAH) is the most common cause of pediatric adrenal insufficiency. Children diagnosed with CAH need exogenous glucocorticoid therapy throughout their lifetime. Because low dose hydrocortisone (HC) tablets are not available, patients and their caregivers must either split/crush hydrocortisone tablets or rely on HC that has been compounded from adult medications. In late 2020, a pediatric specific HC formulation, an oral granule, became available with doses as low as 0.5 mg. There are minimal data concerning the long-term use of this novel HC formulation. Subject and Method: This case study is a girl (DOB 11/2016) with CAH who has been administered the HC oral granule formulation for over 3 years. Results: The subject is a 7-year-old female who was diagnosed at birth with CAH. At an outside institution, she administered 6.25 mg HC, divided into three doses of 2.5 mg in the morning, 1.25 mg in the afternoon, and 2.5 mg in the evening (2.5/1.25/2.5; 9 mg/m2). She was also administered 0.15 mg fludrocortisone qd. Her dose was increased to a total dose of 7.5 mg, administered 2.5 mg three times/day (10 mg/m2) because of a high 17-OHP concentration, 1048 mg/dL. Other hormone levels were unremarkable. Her 17-OHP levels increased to 2662 mg/dL after 3 months of the increased dose. Because of the development of Cushing’s symptoms, she was switched to the 7.0 mg HC granules (2.5/2.0/2.5; 9.5 mg/m2 total dose). After the switch to the HC granules, her 17-OHP levels was 293 ng/dL. Because of rising 17-OHP levels, her dose needed to be increased twice over the past 2 yrs. Her current dose is 12 mg HC granules (4/4/4; 11 mg/m2 total dose), and her three most recent 17-OHP levels were 73, 101, and 89 ng/dL, respectively. She is also taking 0.2 mg fludrocortisone po, q am. Throughout HC granule therapy, she had no incidence of adrenal crisis. Her most recent calculated bone age was 8 yr 10 mo, with her chronological age was 6 yr, 8 mo with no evidence of virilization. Growth curves suggest that she is neither under- nor over-dosed on her HC therapy. Conclusion: This case may be the longest, continuous administration of the novel HC granules in US for a child with CAH. The results show the potential to better titrate HC dosing, especially with the narrow therapeutic window of HC in children with CAH. Her daily dose was adjusted by titrating in small increments to result in good outcomes, such as better hormonal control, the lack of virilization and continuous growth. Further evaluations of the long-term use of HC granules are needed to occur to establish the long-term risks and benefits of this novel HC granule. Presentation: 6/3/2024

Aliphatic glucosinolates are compounds which occur in high concentrations in Arabidopsis thaliana and other Brassicaceae species. They are important for the resistance of the plant to pest insects. Previously, the biosynthesis of these compounds was shown to be regulated by transcription factors MYB28 and MYB29. We now show that MYB28 and MYB29 are partially redundant, but in the absence of both, the synthesis of all aliphatic glucosinolates is blocked. Untargeted and targeted biochemical analyses of leaf metabolites showed that differences between single and double knock-out mutants and wild type plants were restricted to glucosinolates. Biosynthesis of long-chain aliphatic glucosinolates was blocked by the myb28 mutation, while short-chain aliphatic glucosinolates were reduced by about 50% in both the myb28 and the myb29 single mutants. Most remarkably, all aliphatic glucosinolates were completely absent in the double mutant. Expression of glucosinolate biosynthetic genes was slightly but significantly reduced by the single myb mutations, while the double mutation resulted in a drastic decrease in expression of these genes. Since the myb28myb29 double mutant is the first Arabidopsis genotype without any aliphatic glucosinolates, we used it to establish the relevance of aliphatic glucosinolate biosynthesis to herbivory by larvae of the lepidopteran insect Mamestra brassicae. Plant damage correlated inversely to the levels of aliphatic glucosinolates observed in those plants: Larval weight gain was 2.6 fold higher on the double myb28myb29 mutant completely lacking aliphatic glucosinolates and 1.8 higher on the single mutants with intermediate levels of aliphatic glucosinolates compared to wild type plants.

The formation of endothelial tight junctions (TJs) is crucial in blood-brain barrier (BBB) differentiation, and the expression and targeting of TJ-associated proteins mark the beginning of BBB functions. Using confocal microscopy, this study analyzed endothelial TJs in adult human cerebral cortex and the fetal telencephalon and leptomeninges in order to compare the localization of two TJ-associated transmembrane proteins, occludin and claudin-5. In the arterioles and microvessels of adult brain, occludin and claudin-5 form continuous bands of endothelial immunoreactivity. During fetal development, occludin and claudin-5 immunoreactivity is first detected as a diffuse labeling of endothelial cytoplasm. Later, at 14 weeks, the immunosignal for both proteins shifts from the cytoplasm to the interface of adjacent endothelial cells, forming a linear, widely discontinuous pattern of immunoreactivity that achieves an adult-like appearance within a few weeks. These results demonstrate that occludin and claudin-5 expression is an early event in human brain development, followed shortly by assembly of both proteins at the junctional areas. This incremental process suggests more rapid establishment of the human BBB, consistent with its specific function of creating a suitable environment for neuron differentiation and neurite outgrowth during neocortical histogenesis.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor working as an endothelial cell-specific mitogen and exerting a trophic effect on neurons and glial cells, both these activities being essential during central nervous system vascularisation, development and repair. The vascularisation of human telencephalon takes place by means of an angiogenic mechanism, which starts at the beginning of corticogenesis and actively proceeds up to the last neuronal migration, when the basic scheme of the vascular network has been drawn. Our study focused on VEGF during this critical developmental period with the aim of identifying the cells that express VEGF and of correlating the events of angiogenesis with the main events of cerebral cortex formation. The results show that in fetal human brain VEGF protein is located on multiple cell types, cells proper to the nervous tissue, neuroepithelial cells, neuroblasts and radial glia cells, and non-neuronal cells, endothelial and periendothelial cells. In these cells VEGF expression appears developmentally regulated and is correlated with angiogenesis, which in turn responds to the high metabolic demands of the differentiating neocortex.

The involvement of the metalloprotease-2 (MMP-2) in vessel development was investigated in the human telencephalon by double immunoreactions with antibodies to the enzyme, latent (proMMP-2) and active (aMMP-2) forms, and an antibody against collagen type IV, a constitutive component of the extracellular matrix (ECM) of the vessel basal lamina. MMP-2 is expressed in both 12- and 18-week telencephalic vessels, the proenzyme being mainly localised in endothelial cells and the active form prevailing in alpha-actin-reactive periendothelial cells identified as pericytes. Endothelial cells intensely positive for aMMP-2 were revealed in some microvessels and appeared locally associated with discontinuities of the collagen basal lamina. No detectable expression of MMP-2 was observed in perivascular glial processes revealed by vimentin/glial fibrillary acidic protein immunostainings. Double immunoreactions performed to further investigate telencephalon angiogenesis have demonstrated that both the endothelial cells and pericytes strongly express vascular endothelial growth factor (VEGF). Taken together, the results indicate that MMP-2 is largely involved in human brain angiogenesis and suggest that endothelial cells and pericytes tightly interplay in both angiogenesis mechanisms, by ECM proteolysis, and angiogenesis regulation, by local (autocrine/juxtacrine) VEGF action.

The distribution of cholinergic nerve fibres associated with the microvasculature of the human parietal cerebral cortex was investigated by immunocytochemistry, employing monoclonal antibodies against choline acetyl-transferase, the acetylcholine-synthesizing enzyme. The results revealed strongly immunoreactive nerve fibres in the tunica adventitia of arterioles penetrating the superficial cortical layers from the pial vasculature. Networks of stained nerve fibres were seen within the tunica muscularis of the radially directed arterioles that cross the intermediate and deep cortical laminae, and of their transverse and recurrent branches. Tiny positive nerve fibres were also seen around the cortex capillaries, some reaching the endothelial cells. The morphological data support the involvement of acetylcholine in microvasculature local regulation, possibly with a differentiated role in the arterioles and capillaries.

Die beiden Angiotensin-Konversionsenzym-Hemmer Cilazapril und Captopril wurden in Konzentrationen von 10 bzw. 100 mg/Tag an spontan hypertensive Ratten (SHR) und an normo-tensive Wistar (W)-Ratten von der 12. zur 22. Lebenswoche ver-abreicht. Für beide Substanzen konnte bei SHR eine statistisch signifikante Reduktion des systolischen und diastolischen Blut-druckes, der linksventrikulären Masse und des Index der links-ventrikulären Hypertrophie nachgewiesen werden. Nach Cila-zaprilbehandlung glich sich die Morphologie der Kardiozyten bei SHR weitgehend denen von unbehandelten normotensiven Ratten an, wohingegen die Myofibrillen von captoprilbehandel-ten Ratten ungeordnet, nicht parallel und schmaler als norma-lerweise vorlagen. Darüber hinaus waren elektronenoptisch transparente Sarkoplasmabereiche zwischen den Myofibrillen-bündeln nachweisbar. Auch die Mitochondrien wiesen Verän-derungen auf. Bei den W-Ratten konnten durch die beiden Substanzen keine statistisch signifikanten Veränderungen des Blutdrucks, des Ventrikelgewichts oder des Hypertrophieindex induziert werden. Allerdings wurden für beide Substanzen unterschiedliche Wirkungen auf die morphologische Ultrastruktur des Myokards bestätigt. Diese Beobachtungen geben Hinweise darauf, dass diese beiden in ihrer molekularen Struktur verschiedenen Substanzen einen unterschiedlichen Einfluss auf Gewebe ausüben, obwohl sie hinsichtlich Blutdruck und links-ventrikulärer Masse ähnliche Effekte auslösen.

The hypothesis of astroglial cell involvement in prenatal setting up of the blood-brain barrier (BBB) has been examined by producing glial degeneration in cerebellum of chicken embryos submitted to the action of gliotoxin 6-aminonicotinamide (6-AN), which was applied onto the embryo chorioallantoic membrane during both early and late embryonic development. The effects of 6-AN on the cerebellum astroglial cells and microvessels were analysed under the light microscope by immunostaining for 3CB2 (chick-specific glial marker) and HT7 (chick-specific marker of BBB-provided brain endothelia), under the electron microscope, as well as by the vascular permeability tracer horseradish peroxidase. The results, showing good suitability of the 6-AN model also when applied in early embryonic development, demonstrated a correlation between perivascular glia depletion and endothelial barrier impairment and suggested that astroglia play a role in the BBB prenatal differentiation.

Connexin43 (Cx43), the main protein constituting the gap junctions between astrocytes, has previously been demonstrated in endothelial cells of somatic vessels where the intercellular coupling that it provides plays a role in endothelial proliferation and migration. In this study, Cx43 expression was analysed in human brain microvascular endothelial cells of the cortical plate of 18-week foetal telencephalon, in adult cerebral cortex and glioma (astrocytomas). The study was carried out by immunocytochemistry utilizing a Cx43 monoclonal antibody and a polyclonal antibody anti-GLUT1 (glucose transporter isoform 1) to identify the endothelial cells and to localize Cx43. Endothelial Cx43 is differently expressed in the cortical plate, cerebral cortex and astrocytoma. Within the cortical plate and tumour, Cx43 is highly expressed in microvascular endothelial cells whereas it is virtually absent in the cerebral cortex microvessels. The high expression of the gap junction protein in developing brain, as well as in brain tumours, may be related to the growth status of the microvessels during brain and tumour angiogenesis. The lack of endothelial Cx43 in the cerebral cortex is in agreement with the characteristics of the mature brain endothelial cells that are sealed by tight junctions. In conclusion, the results indicate that endothelial Cx43 expression is developmentally regulated in the normal human brain and suggest that it is controlled by the microenvironment in both normal and tumour-related conditions.[PUBLICATION ABSTRACT]