Brain edema is detrimental in ischemic stroke and its treatment options are limited. Kinins are proinflammatory peptides that are released during tissue injury. The effects of kinins are mediated by ...2 different receptors (B1 and B2 receptor B1R and B2R) and comprise induction of edema formation and release of proinflammatory mediators.
Focal cerebral ischemia was induced in B1R knockout, B2R knockout, and wild-type mice by transient middle cerebral artery occlusion. Infarct volumes were measured by planimetry. Evan's blue tracer was applied to determine the extent of brain edema. Postischemic inflammation was assessed by real-time reverse-transcriptase polymerase chain reaction and immunohistochemistry. To analyze the effect of a pharmacological kinin receptor blockade, B1R and B2R inhibitors were injected.
B1R knockout mice developed significantly smaller brain infarctions and less neurological deficits compared to wild-type controls (16.8+/-4.7 mm(3) vs 50.1+/-9.1 mm(3), respectively; P<0.0001). This was accompanied by a dramatic reduction of brain edema and endothelin-1 expression, as well as less postischemic inflammation. Pharmacological blockade of B1R likewise salvaged ischemic tissue (15.0+/-9.5 mm(3) vs 50.1+/-9.1 mm(3), respectively; P<0.01) in a dose-dependent manner, even when B1R inhibitor was applied 1 hour after transient middle cerebral artery occlusion. In contrast, B2R deficiency did not confer neuroprotection and had no effect on the development of tissue edema.
These data demonstrate that blocking of B1R can diminish brain infarction and edema formation in mice and may open new avenues for acute stroke treatment in humans.
Kinins are important mediators in cardiovascular homeostasis, inflammation, and nociception. Two kinin receptors have been described, B1 and B2. The B2 receptor is constitutively expressed, and its ...targeted disruption leads to salt-sensitive hypertension and altered nociception. The B1 receptor is a heptahelical receptor distinct from the B2 receptor in that it is highly inducible by inflammatory mediators such as bacterial lipopolysaccharide and interleukins. To clarify its physiological function, we have generated mice with a targeted deletion of the gene for the B1 receptor. B1 receptor-deficient animals are healthy, fertile, and normotensive. In these mice, bacterial lipopolysaccharide-induced hypotension is blunted, and there is a reduced accumulation of polymorphonuclear leukocytes in inflamed tissue. Moreover, under normal noninflamed conditions, they are analgesic in behavioral tests of chemical and thermal nociception. Using whole-cell patch-clamp recordings, we show that the B1 receptor was not necessary for regulating the noxious heat sensitivity of isolated nociceptors. However, by using an in vitro preparation, we could show that functional B1 receptors are present in the spinal cord, and their activation can facilitate a nociceptive reflex. Furthermore, in B1 receptor-deficient mice, we observed a reduction in the activity-dependent facilitation (wind-up) of a nociceptive spinal reflex. Thus, the kinin B1 receptor plays an essential physiological role in the initiation of inflammatory responses and the modulation of spinal cord plasticity that underlies the central component of pain. The B1 receptor therefore represents a useful pharmacological target especially for the treatment of inflammatory disorders and pain.
Transgenic and gene-targeting technologies allowing the generation of genetically altered animal models have greatly advanced our understanding of the function of specific genes. This is also true ...for the kallikrein-kinin system (KKS), in which some, but not yet all, components have been functionally characterized using such techniques. The first genetically altered animal model for a KKS component was supplied by nature, the brown Norway rat carrying an inactivating mutation in the kininogen gene. Mice deficient in tissue kallikrein, B1 and B2 receptors, some kinin-degrading enzymes, and factor XII followed, together with transgenic rat and mouse strains overexpressing tissue kallikrein, B1 and B2 receptors, and degrading enzymes. There are still no animal models with genetic alterations in plasma kallikrein, kininases I and some other degrading enzymes. The models have confirmed an important role of the KKS in cardiovascular pathology, inflammation, and pain, and have partially elucidated the distinct function of the two receptors. This created the basis for rational decisions concerning the putative use of kinin receptor agonists and antagonists in therapeutic applications. However, a more thorough analysis of the existing models and the generation of new, more sophisticated transgenic models will be necessary to clarify the still elusive issue as to where and by which mechanisms the kinins exert their actions.
Aims: To evaluate the expression of neutrophilic cathelicidin pro-LL-37 (hCAP18) and the expression of CAMP gene in patients with chronic primary neutropenia (CPN), and its correlation with the ...etiology of the disease. Methods: Prospective study, carried out from 2021 to 2023, including children and adult patients treated at a neutropenia reference service. We analyzed 4 patients with severe congenital neutropenia (SCN), 2 with cyclic neutropenia (CN), 3 with autoimmune neutropenia (AIN), 3 with chronic idiopathic neutropenia (CIN) and 2 patients with glycogen storage disease 1B (DDG1B). The hCAP18 protein expression was evaluated in the plasma of patients with different types of neutropenia and healthy control individuals, using the Western-Blot technique. The CAMP gene expression assay was performed using the real-time PCR (qPCR) technique, using RNA extracted from whole blood. Results: The expression of hCAP18 was not observed in patients with SCN, even after increasing the neutrophil count using filgrastim. In individuals with CN, the expression of hCAP18 was also not observed during the neutropenic phase, but the protein was detected in the phase of normalization of the neutrophil count, showing a positive correlation of the protein expression with the number of neutrophils in this etiology, different from that of SCN. Expression of hCAP18 was also detected in patients with AIN, CIN and DDG1B, despite pronounced neutropenia in some patients, and in the control group. The qPCR showed a low expression of CAMP in patients with SCN, while the other etiologies showed variations in gene expression, being positive in some individuals and negative in others with the same etiology. However, using this method, it was possible to notice a positive correlation (r = 0.732 and p < 0.05 – Spearman test) between the expression of the CAMP gene and the expression of hCAP18. Discussion: hCAP18, encoded by the CAMP gene, is an important component of the secondary granules of neutrophils, with immunological and bactericidal activity. The results of this work corroborate the data by Karlsson et al., who showed absence or significant decrease in the expression of hCAP18 in individuals with SCN and its expression in individuals with CIN and AIN. In addition, we also demonstrated a positive correlation between the expression of the CAMP gene and hCAP18 and a decrease in the expression of the CAMP gene in patients with SCN. Therefore, plasma hCAP18 assessment is suggested as a differential CPN screening method, aiding in the diagnosis of SCN due to low or undetectable hCAP18 levels. Conclusion: There is a correlation between CAMP gene expression and hCAP18 expression with CPN. In addition, we also demonstrated that hCAP18 is not expressed in patients with SCN and in the neutropenic phase of CN. Thus, the use of this technique is suggested as a new screening routine for the investigation of patients with CPN, especially in patients with SCN and CN, differentiating them from the others and avoiding more invasive tests.
The kinin receptors are classically involved in inflammation, pain and sepsis. The effects of the kinin B1 receptor agonist des-Arg9-bradykinin (DBK) and lipopolysaccharide (LPS) were investigated by ...comparing the membrane potential responses of aortic rings from transgenic rats overexpressing the kinin B1 receptor (B1R) in the endothelium (TGR(Tie2B1)) and Sprague Dawley (SD) rats. No difference in the resting membrane potential in the aorta’s smooth muscle from the transgenic and SD rats was observed. The aorta rings from SD rats hyperpolarized only to LPS but not to DBK, whereas the aorta rings from TGR(Tie2B1) responded by the administration of both drugs. DBK and LPS responses were inhibited by the B1 receptor antagonist R715 and by iberiotoxin in both cases. Thapsigargin induced a hyperpolarization in the smooth muscle of SD rats that was not reversed by R715, but was reversed by iberiotoxin and this hyperpolarization was further augmented by DBK administration. These results show that the model of overexpression of vascular B1 receptors in the TGR(Tie2B1) rats represent a good model to study the role of functional B1 receptors in the absence of any pathological stimulus. The data also show that KCa channels are the final mediators of the hyperpolarizing responses to DBK and LPS. In addition, we suggest an interaction between the B1R and TLR4, since the hyperpolarization induced by LPS could be abolished in the presence of R715.
It has been described recently that the nonpeptide AVE 0991 (AVE) mimics the effects of angiotensin-(1-7) Ang-(1-7) in bovine endothelial cells. In this study, we tested the possibility that AVE is ...an agonist of the Ang-(1-7) receptor Mas, in vitro and in vivo. In water-loaded C57BL/6 mice, AVE (0.58 nmol/g body weight) produced a significant reduction in urinary volume (0.06+/-0.03 mL/60 min n=9 versus 0.27+/-0.05 n=9; P<0.01), associated with an increase in urinary osmolality. The Ang-(1-7) antagonist A-779 completely blocked the antidiuretic effect of AVE. As observed previously for Ang-(1-7), the antidiuretic effect of AVE after water load was blunted in Mas-knockout mice (0.37+/-0.10 mL/60 min n=9 versus 0.27+/-0.03 mL/60 min n=11 AVE-treated mice). In vitro receptor autoradiography in C57BL/6 mice showed that the specific binding of 125I-Ang-(1-7) to mouse kidney slices was displaced by AVE, whereas no effects were observed in the binding of 125I-angiotensin II or 125I-angiotensin IV. Furthermore, AVE displaced the binding of 125I-Ang-(1-7) in Mas-transfected monkey kidney cells (COS) cells (IC50=4.75x10(-8) mol/L) and of rhodamine-Ang-(1-7) in Mas-transfected Chinese hamster ovary (CHO) cells. It also produced NO release in Mas-transfected CHO cells blocked by A-779 but not by angiotensin II type-1 (AT1) and AT2 antagonists. Contrasting with these data, the antidiuretic effect of AVE was totally blocked by AT2 antagonists and partially blocked (approximately 60%) by AT1 antagonists. The binding data, the results obtained in Mas-knockout mice and in Mas-transfected cells, show that AVE is a Mas receptor agonist. Our data also suggest the involvement of AT2/AT1-related mechanisms, including functional antagonism, oligomerization or cross-talk, in the renal responses to AVE.
To describe 2 cases of autoimmune neutropenia (AIN) patients infected with Sars-Cov-2.
Two subjects case report.
Case report 1: A girl with primary AIN since 1 year and 10 months old, maintaining ...severe neutropenia and mild recurrent infections. Presented to the emergency department in June/2020, at 3 years and 8 months old, with flu-like symptoms, afebrile, in good general condition. Physical examination was normal. The absolute neutrophil count (ANC) was 0.279 × 109/L. At hospital admission, Sars-Cov-2 (RT-PCR) tested positive and filgrastim (G-CSF) 5 μg/kg/day was initiated. Chest X-ray was also normal and blood culture resulted negative. She remained in great general condition, afebrile, and was discharged on the 2nd day of hospitalization, with clarithromycin (15 mg/kg/day). After G-CSF, ANC: 0.494 × 109/L (1st dose), 1.431 × 109/L (2nd dose). On outpatient follow-up, she had no long-term complications from Covid-19. Case Report 2: A man with chronic immune thrombocytopenia purpura (ITP) since 2008, autoimmune hemolytic anemia since 2013, evolved with AIN on May/2020, at 42 years old, with ANC lower than 0.5 × 109/L. On 6/1/2020, he had ANC 0.170 × 109/L. On 6/25/2020, he started flu-like symptoms, had ANC of 5.118 × 109/L, and tested positive for Sars-Cov-2 (RT-PCR). He kept high fever (102,2°F) and was hospitalized for 10 days without use of G-CSF. After discharge, on outpatient follow-up, he had no long-term complications from Covid-19, and presented ANC 0.338 × 109/L (Aug/2020).
At beginning of Covid-19 pandemic, severity infection in children was unknown. Today is known that most of them have milder clinical course, regardless of chronic diseases. In adults, in contrast, the inflammatory response tends to exacerbation, with more severe clinical conditions. Furthermore, many case reports of patients infected by SARS-CoV-2 with comorbidities literature are published. However, to date there are no reports on the impact of COVID-19 in AIN patients. Increased neutrophil counts during infectious episodes are common in AIN, which appears to be related to the benign course of most infections. We reported 2 cases of AIN patients diagnosed with Covid-19, both with favorable clinical outcomes despite heterogenic clinical course. On the first case, she presented few symptoms and ANC increased only after using G-CSF. On the second reported patient, there was a spontaneous increase of ANC and greater inflammatory response than the first case. It could suggest a correlation between inflammatory response to COVID-19 and ANC in cases of autoimmune neutropenia.
In the reported cases, clinical course of disease and neutrophil count were different between adult and pediatric patients. It is not possible to state whether this difference is due to age group, individual response to infection or other variables. It is important to assess other cases of AIN infected by COVID 19 to better understand correlation between severity of infection and neutrophil count response.
To describe a child carrying a GFI1 -variant with autoimmune neutropenia.
Single subject case report.
This is a case report of a 1 year-and-10-months-old female presenting severe chronic neutropenia ...(0,03 × 109/L) with mild infections. The frequency of infections reduced as she got older. Physical examinations outside of infections were always normal and she had expected development. She did not have monocytosis or other hematologic disorders, and lymphocytes evaluation by flow cytometry analysis resulted normal. Viral diseases were negative, immunoglobulins dosage was normal for the age group, and screening for systemic autoimmune disease (sAID) was negative. Bone marrow aspirate analysis with karyotype resulted normal, without maturation arrest or dysplasia. Inborn errors of metabolism (IRM) and immunity (IRI) were ruled out. Antineutrophil (anti-HNA) autoantibody was detected on two occasions: at ages of 2-years-and-9-months-old and 5-years-and-3-months-old, presenting the same intensity and the same pan-reactivity to the FCγRIIIb glycoprotein. She has no siblings, but her father presented recurrent otitis until 6-year-old and hemogram with a persistent mild neutropenia documented since childhood. Ethnic benign neutropenia was ruled out. Both expressed Fyb on erythrocytes and were heterozygotes for single nucleotide polymorphism rs2814778 in the ACKR1. She did not present remission of the disease until the age of 5 years and 7 months. A whole exome sequencing, showed a heterozygotic non-pathogenic GFI-1 variant in exon 6 (c.929G>A:p.Arg310Gln). The variant, confirmed by Sanger sequencing, was also present in her father. He has no antineutrophil antibodies detected to date.
Most cases of pAIN are diagnosed between 3 to 8 months. Spontaneous remission in about 90% could be expected in 7 to 24 months of diagnosis or up to age of 4-5 years. In this reported case, the patient had severe neutropenia demonstrated since 1 year-and-10-month-old. Even 45 months after the beginning she remains neutropenic, carrying anti-HNA autoantibody with the same pan-reactivity features. The persistence of anti-HNA with no specificity may be associated to persistent autoimmune neutropenia (AIN) as well as a characteristic of secondary AIN, but screening for both sAID and IEI were negative. Due to the persistence of neutropenia, a whole exome sequencing was performed, presenting GFI-1 variant inherited from her father, also neutropenic. The patient does not present features of GFI-1 -variants, clinical course and the bone marrow were not compatible with SCN. In addition to regulating myelopoiesis, GFI-1 also plays a role in the maturation of B and T lymphocytes, and in process of antigen-mediated lymphocyte activation. Although mature B-cells do not express GFI-1, this loss may be correlated with the increase of some IgG fractions, and some works have correlated GFI-1 changes with autoimmune conditions and could be the cause of AIN reported.
For patients with persistent pAIN, perfoming tests to detect a genetic involvement may be useful to understand the cause of the disease. In this case, the presence of GFI-1 variant could be the trigger of AIN reported.
AN UNUSUAL CASE OF NEUTROPENIA Franco, JFS; Souza, RC; Franco, JM ...
Hematology, Transfusion and Cell Therapy,
October 2022, 2022-10-00, Letnik:
44
Journal Article
Recenzirano
Odprti dostop
To describe a child with neutropenia and carrying inborn error of metabolism (IEM).
A 10 months-old boy, fist child of non-consanguineous parents, presented severe neutropenia (0.02 × 109/L) with ...fever, feeding problems, hypertonia and seizures. The frequency of fever and oscillating neutropenia is raised beyond use of antibiotics and granulocyte colony-stimulating factors. Some facial features present during physical examinations (hypertelorism, coarse face and skin dyspigmentation). He has failure to thrive and neuropsychomotor developmental delay. Viral diseases, organic acid in urine and mass spectrometry in tandem was normal. Abdominal ultrasound showed right hydronephrosis. Other image exams not detected alterations. The bone marrow aspirate analysis with karyotype resulted normal, with mild maturation arrest of granulocytic series. He did not have monocytosis or other hematologic disorders. Serum immunoglobulins were normal for the age group. Viral chronic diseases were negative, and she had no other autoimmune disorders. His mother had a first trimester spontaneous abortion, his father has no illnesses, and he has no siblings. A whole exome NGS sequencing showed compound heterozygous: one missense pathogenic CLPB variant (p. Arg408Gly) and other VUS c.1770+2T>A, splicing site. Hospitalizations is still frequent, and neutropenia did not remit to date.
This patient presents neutropenia and neurologic involvement, characteristics of caseinolytic peptidase B (CLPB) deficiency, which can range from severe to mild. In this reported case, a severe CLPB deficiency, death usually occurs at a few months of age due to neonatal neurologic involvement (hypotonia or hypertonia, respiratory insufficiency, swallowing problems, absence of voluntary movements, and epilepsy) and severe neutropenia associated with life-threatening infections. A multidisciplinary team is necessary, and no specific dietary or metabolic treatment is available. Hematopoietic stem cell transplant is not indicated in this case. Granulocyte-colony stimulating factor to increase neutrophil counts to reduce the frequency of infections should be used. Neutropenia associated with CLPB disease is present from birth and may be chronic or intermittent with absolute neutrophil counts ranging from severe (< 0.5 per mm3) to mild (< 1.5 per mm3). Elevated urinary excretion of 3-methylglutaconic acid (3-MGA) (typically 2x-10x higher than the reference range) has been observed in most of affected individuals to date.
This case may be useful to understand the neutropenia in inborn error of metabolism and clinical signs and symptoms of the disease. The molecular evaluation is fundamental to elucidate diagnostic of neutropenia in newborns.
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