Results of Complex Immunopharmacological Analysis of the Mechanisms of Action of Bioregulation Agents.

Elucidation of the pharmacodynamic mechanisms of drugs capable of potentiating the effects of non-steroidal anti-inflammatory drugs is an important task. In this in vitro study, the ability of Traumeel S to influence the innate and acquired immunity was evaluated. Traumeel S was found to reduce activities of NADPH oxidase and neutrophil extracellular traps, as well as to evoke anti-inflammatory activity of lymphocyte subpopulations.

Pathological and immunological characterization of bluetongue virus serotype 1 infection in type I interferons blocked immunocompetent adult mice.

Introduction: Wild-type adult mice with intact interferon (IFN) system were neither susceptible to bluetongue virus (BTV) infection nor showed signs of morbidity/mortality. Establishment of immunologically competent wild-type adult mouse model with type I IFNs blockade is necessary to assess the pathogenesis, immune responses and testing of BTV vaccines. Objectives: Present study aimed to establish and characterize BTV serotype 1 infection in immunocompetent adult mice with type I IFNs blockade at the time of infection by studying immune responses and sequential pathology. Methods: Adult mice were administered with anti-mouse IFN-α/ß receptor subunit-1 (IFNAR1) blocking antibody (Clone: MAR1-5A3) 24 h before and after BTV serotype 1 infection, and sacrificed at various time points. Sequential pathology, BTV localization by immunohistochemistry and quantification by qRT-PCR, immune cell kinetics and apoptosis by flow cytometry, and cytokines estimation by c-ELISA and qRT-PCR were studied. Results: IFNAR blocked-infected mice developed clinical signs and typical lesions of BT; whereas, isotype-infected control mice did not develop any disease. The IFNAR blocked-infected mice showed enlarged, edematous, and congested lymph nodes (LNs) and spleen, and vascular (congestion and hemorrhage) and pneumonic lesions in lungs. Histopathologically, marked lymphoid depletion with "starry-sky pattern" due to lymphocytes apoptosis was noticed in the LNs and spleen. BTV antigen was detected and quantified in lymphoid organs, lungs, and other organs at various time points. Initial leukopenia (increased CD4+/CD8+ T cells ratio) followed by leukocytosis (decreased CD4+/CD8+ T cells ratio) and significantly increased biochemical values were noticed in IFNAR blocked-infected mice. Increased apoptotic cells in PBMCs and tissues coincided with viral load and levels of different cytokines in blood, spleen and draining LNs and notably varied between time points in IFNAR blocked-infected mice. Conclusion: Present study is first to characterize BTV serotype 1 infection in immunocompetent adult mouse with type I IFNs blockade. The findings will be useful for studying pathogenesis and testing the efficacy of BTV vaccines.

A homozygous stop-gain variant in ARHGAP42 is associated with childhood interstitial lung disease, systemic hypertension, and immunological findings.

ARHGAP42 encodes Rho GTPase activating protein 42 that belongs to a member of the GTPase Regulator Associated with Focal Adhesion Kinase (GRAF) family. ARHGAP42 is involved in blood pressure control by regulating vascular tone. Despite these findings, disorders of human variants in the coding part of ARHGAP42 have not been reported. Here, we describe an 8-year-old girl with childhood interstitial lung disease (chILD), systemic hypertension, and immunological findings who carries a homozygous stop-gain variant (c.469G>T, p.(Glu157Ter)) in the ARHGAP42 gene. The family history is notable for both parents with hypertension. Histopathological examination of the proband lung biopsy showed increased mural smooth muscle in small airways and alveolar septa, and concentric medial hypertrophy in pulmonary arteries. ARHGAP42 stop-gain variant in the proband leads to exon 5 skipping, and reduced ARHGAP42 levels, which was associated with enhanced RhoA and Cdc42 expression. This is the first report linking a homozygous stop-gain variant in ARHGAP42 with a chILD disorder, systemic hypertension, and immunological findings in human patient. Evidence of smooth muscle hypertrophy on lung biopsy and an increase in RhoA/ROCK signaling in patient cells suggests the potential mechanistic link between ARHGAP42 deficiency and the development of chILD disorder.

Prognostic implications of neutrophil-lymphocyte ratio in COVID-19.

BACKGROUND: The clinical presentation of COVID-19 ranges from a mild, self-limiting disease, to multiple organ failure and death. Most severe COVID-19 cases present low lymphocytes counts and high leukocytes counts, and accumulated evidence suggests that in a subgroup of patients presenting severe COVID-19, there may be a hyperinflammatory response driving a severe hypercytokinaemia which may be, at least in part, signalling the presence of an underlying endothelial dysfunction. In this context, available data suggest a prognostic role of neutrophil-lymphocyte ratio (NLR) in various inflammatory diseases and oncological processes. Following this rationale, we hypothesized that NLR, as a marker of endothelial dysfunction, may be useful in identifying patients with a poor prognosis in hospitalized COVID-19 cases. DESIGN: A retrospective observational study performed at Hospital Universitario HM Puerta del Sur, Madrid, Spain, which included 119 patients with COVID-19 from 1 March to 31 March 2020. Patients were categorized according to WHO R&D Expert Group. RESULTS: Forty-five (12.1%) patients experienced severe acute respiratory failure requiring respiratory support. Forty-seven (12.6%) patients died. Those with worse outcomes were older (P = .002) and presented significantly higher NLR at admission (P = .001), greater increase in Peak NLR (P < .001) and higher increasing speed of NLR (P = .003) compared with follow-up patients. In a multivariable logistic regression, age, cardiovascular disease and C-reactive protein at admission and Peak NLR were significantly associated with death. CONCLUSIONS: NLR is an easily measurable, available, cost-effective and reliable parameter, which continuous monitoring could be useful for the diagnosis and treatment of COVID-19.

Bivalent Inhibitor with Selectivity for Trimeric MMP-9 Amplifies Neutrophil Chemotaxis and Enables Functional Studies on MMP-9 Proteoforms.

A fundamental part of the immune response to infection or injury is leukocyte migration. Matrix metalloproteinases (MMPs) are a class of secreted or cell-bound endopeptidases, implicated in every step of the process of inflammatory cell migration. Hence, specific inhibition of MMPs is an interesting approach to control inflammation. We evaluated the potential of a bivalent carboxylate inhibitor to selectively inhibit the trimeric proteoform of MMP-9 and compared this with a corresponding monovalent inhibitor. The bivalent inhibitor efficiently inhibited trimeric MMP-9 (IC50 = 0.1 nM), with at least 500-fold selectivity for MMP-9 trimers over monomers. Surprisingly, in a mouse model for chemotaxis, the bivalent inhibitor amplified leukocyte influxes towards lipopolysaccharide-induced inflammation. We verified by microscopic and flow cytometry analysis increased amounts of neutrophils. In a mouse model for endotoxin shock, mice treated with the bivalent inhibitor had significantly increased levels of MMP-9 in plasma and lungs, indicative for increased inflammation. In conclusion, we propose a new role for MMP-9 trimers in tempering excessive neutrophil migration. In addition, we have identified a small molecule inhibitor with a high selectivity for the trimeric proteoform of MMP-9, which will allow further research on the functions of MMP-9 proteoforms.

West Nile virus neuroinvasive disease associated with rituximab therapy.

West Nile virus neuroinvasive disease (WNVND) manifests with meningitis, encephalitis, and/or acute flaccid paralysis. It represents less than 1% of the clinical syndromes associated with West Nile virus (WNV) infection in immunocompetent patients. Immunosuppressive therapy is associated with increased risk of WNVND and worse prognosis. We present a patient with WNVND during therapy with rituximab, and a review of the literature for previous similar cases with the goal to describe the clinical spectrum of WNVND in patients treated specifically with rituximab. Our review indicates that the most common initial complaints are fever and altered mental status, brain magnetic resonance imaging often shows bilateral thalamic hyperintensities, and cerebrospinal analysis consistently reveals mild lymphocytic pleocytosis with elevated protein, positive WNV polymerase chain reaction, and negative WNV antibodies. Treatment is usually supportive care, with intravenous immunoglobulins (IVIG) plus corticosteroids and WNV-specific IVIG also used. The disease is usually fatal despite intervention. Our patient's presentation was very similar to prior reports, however demonstrated spontaneous improvement with supportive management only. WNVND is a rare and serious infection with poor prognosis when associated with rituximab therapy. Diagnosis is complicated by absent or delayed development of antibodies. The presence of bilateral thalamic involvement is a diagnostic clue for WNVND. There is insufficient evidence to recommend the use of corticosteroids or IVIG.

Beyond LNB: Real life data on occurrence and extent of CSF CXCL13 in neuroinflammatory diseases.

To evaluate occurrence and extent of CSF CXCL13 elevations beyond Lyme neuroborreliosis, we investigated CXCL13 in an unselected patient cohort with neuroinflammatory disease. From March 2016 to March 2017, 180 in-patients with CSF pleocytosis were categorized into following groups: pyogenic CNS infections, aseptic meningoencephalitis, neuroimmunological diseases, and reactive pleocytosis. We provide evidence that CXCL13 elevation occurs at variable extent in the majority of neuroinflammatory diseases. The exact role of CXCL13 in CSF is elusive, but the broad occurrence in neuroinflammation points at CNS immune activation, which is not exclusive for LNB.

Biased S1PR1 Signaling in B Cells Subverts Responses to Homeostatic Chemokines, Severely Disorganizing Lymphoid Organ Architecture.

Ligand-engaged chemoattractant receptors trigger Gαi subunit nucleotide exchange, stimulating the activation of downstream effector molecules. Activated chemoattractant receptors also dock G protein-coupled receptor kinases (GRKs) that help mediate receptor desensitization. In this study, we show that the B cell-specific loss of GRK2 severely disrupts B cell trafficking and immune cell homeostasis. The GRK2 deficiency in developing murine B cells leads to a severe immune phenotype, including a major reduction of bone marrow IgD+ cells, splenomegaly with a loss of white pulp and grossly expanded red pulp, a deficit of Peyer patches, and small lymph nodes with marked reductions in B cell numbers. The major phenotypes in these mice arise from excessive S1PR1 signaling combined with inadequate homeostatic chemokine receptor signaling. CXCL13 signaling is the most severely compromised. In B cells, our data also indicate that S1PR1 signals constitutively, as blocking S1PR1 signaling with an S1PR1 antagonist enhanced CXCL13-triggered wild-type B cell migration. Furthermore, blocking S1PR1 signaling in the GRK2-deficient B cells partially corrected their poor response to chemokines. Treating mice lacking GRK2 expression in their B cells with an S1PR1 antagonist partially normalized B cell trafficking into lymph node and splenic follicles. These findings reveal the critical interdependence of Gαi-linked signaling pathways in controlling B lymphocyte trafficking.

The E3 ubiquitin ligase SPOP controls resolution of systemic inflammation by triggering MYD88 degradation.

The response to systemic infection and injury requires the rapid adaptation of hematopoietic stem cells (HSCs), which proliferate and divert their differentiation toward the myeloid lineage. Significant interest has emerged in understanding the signals that trigger the emergency hematopoietic program. However, the mechanisms that halt this response of HSCs, which is critical to restore homeostasis, remain unknown. Here we reveal that the E3 ubiquitin ligase Speckle-type BTB-POZ protein (SPOP) restrains the inflammatory activation of HSCs. In the absence of Spop, systemic inflammation proceeded in an unresolved manner, and the sustained response in the HSCs resulted in a lethal phenotype reminiscent of hyper-inflammatory syndrome or sepsis. Our proteomic studies decipher that SPOP restricted inflammation by ubiquitinating the innate signal transducer myeloid differentiation primary response protein 88 (MYD88). These findings unearth an HSC-intrinsic post-translational mechanism that is essential for reestablishing homeostasis after emergency hematopoiesis.

Antitumoral effect of maintained neutrophilia induced by rhG-CSF in a murine model of pancreatic cancer.

Although the protumoral functions of polymorphonuclear neutrophils are well known, some now-forgotten studies report antitumoral roles for these cells. The present work examines the antitumoral effect of maintained neutrophilia induced via the injection of recombinant human granulocyte colony stimulating factor (rhG-CSF, 100 µg/kg/day) in a Panc-1 subcutaneous xenograft murine model of pancreatic cancer. This treatment was compared with gemcitabine administration (120 mg/kg every two days) and a saline control (n = 6-7 mice per group). Compared to the controls, both the rhG-CSF- and gemcitabine-treated mice showed significantly suppressed tumor growth by day 4 (p < 0.001 and p = 0.013 respectively). From a mean starting volume of 106.9 ± 3.1 mm3 for all treatment groups, the final mean tumor volumes reached were 282.0 ± 30.7 mm3 for the rhG-CSF-treated mice, 202.6 ± 18.1 mm3 for the gemcitabine-treated mice and 519.4 ± 62.9 mm3 for the control mice (p < 0.004 and p < 0.01, respectively, vs. control). The rhG-CSF-treated tumors showed higher percentage necrosis than those treated with gemcitabine (37.4 ± 4.6 vs. 7.5 ± 3.0; p < 0.001). This is the first report of a clear anti-tumoral effect of rhG-CSF when used in monotherapy against pancreatic cancer. Since rhG-CSF administration is known to be associated with very few adverse events, it may offer an attractive alternative in the clinical treatment of pancreatic cancer.

Guillain-Barré syndrome with exaggerated pleocytosis and anti-GM1 ganglioside antibodies.

An 81-year-old man presented with fever, confusion and rapidly-progressive flaccid tetraparesis. Clinical presentation and neurophysiology were consistent with a severe axonal polyneuropathy. Anti-GM1 and Campylobacter serology were both positive, consistent with postinfectious axonal-variant Guillain-Barré syndrome (GBS). GBS is characterised by albuminocytological dissociation, where an elevated protein and acellular cerebrospinal fluid are typical. However, in this case, CSF analysis revealed an exaggerated pleocytosis (72 white blood cells (WBC)/mm3). No source of central nervous system infection or inflammation was identified despite thorough investigation. The patient was treated with intravenous immunoglobulin and intensive rehabilitation.Albuminocytological dissociation classically distinguishes GBS from infective causes of flaccid weakness (eg, enteroviruses, flaviviruses and HIV). Diagnostic criteria frequently cite a pleocytosis of <50 WBC/mm3 as required in the diagnosis of GBS. However, this case demonstrates that pleocytosis exceeding this level can occur in the presence of convincing evidence of GBS and without demonstrable neurotropic infection.

Splenectomy-Induced Leukocytosis Promotes Intratumoral Accumulation of Myeloid-Derived Suppressor Cells, Angiogenesis and Metastasis.

BACKGROUND: Enlargement of the spleen is commonly observed in animal models of cancer. Here, in a breast cancer model, it was aimed to determine the effect of splenectomy on circulating and tumor-infiltrating myeloid-derived suppressor cells (MDSCs), tumor angiogenesis, and metastasis. METHODS: Mice were inoculated with 4T1 breast cancer cells and underwent splenectomy or sham laparotomy. Tumor growth and survival of animals were followed. Macroscopic and histopathological analyses were performed to determine splenomegaly and metastasis. Immunophenotyping of myeloid cells was performed with flow cytometric analysis of CD11b, Gr-1, F4/80, CD206, CD11c, and F4/80 markers. Suppressive function of MDSCs on T cell proliferation was studied in cocultures. Tumor angiogenesis and granulocytic myeloid cell infiltration in the metastatic foci were studied by CD31 and Ly6G immunohistochemistry, respectively. RESULTS: The mice bearing breast tumors underwent total splenectomy at an early time point of tumorigenesis when only low levels of MDSCs had accumulated in the spleen. Circulating and tumor-infiltrating MDSCs, and tumor-associated macrophages (TAMs) were increased following splenectomy. Nevertheless, splenectomy could only lead to a temporary deceleration in tumor growth but favored lung metastasis and angiogenesis in the long run. CONCLUSION: Our data demonstrated a link among splenectomy-induced leukocytosis, accumulation of circulating and tumor-infiltrating MDSC, and enhanced angiogenesis and metastasis. Therefore, as a part of oncological surgery, favorable and unfavorable facets of the splenectomy must be considered to improve therapeutic efficacy.

Fatal Pertussis in the Neonatal Mouse Model Is Associated with Pertussis Toxin-Mediated Pathology beyond the Airways.

In infants, Bordetella pertussis can cause severe disease, manifested as pronounced leukocytosis, pulmonary hypertension, and even death. The exact cause of death remains unknown, and no effective therapies for treating fulminant pertussis exist. In this study, a neonatal mouse model of critical pertussis is characterized, and a central role for pertussis toxin (PT) is described. PT promoted colonization, leukocytosis, T cell phenotypic changes, systemic pathology, and death in neonatal but not adult mice. Surprisingly, PT inhibited lung inflammatory pathology in neonates, a result which contrasts dramatically with observed PT-promoted pathology in adult mice. Infection with a PT-deficient strain induced severe pulmonary inflammation but not mortality in neonatal mice, suggesting that death in these mice was not associated with impaired lung function. Dissemination of infection beyond the lungs was also detected in neonatal mice, which may contribute to the observed systemic effects of PT. We propose that it is the systemic activity of pertussis toxin and not pulmonary pathology that promotes mortality in critical pertussis. In addition, we observed transmission of infection between neonatal mice, the first report of B. pertussis transmission in mice. This model will be a valuable tool to investigate causes of pertussis pathogenesis and identify potential therapies for critical pertussis.

Prehospital immune responses and development of multiple organ dysfunction syndrome following traumatic injury: A prospective cohort study.

BACKGROUND: Almost all studies that have investigated the immune response to trauma have analysed blood samples acquired post-hospital admission. Thus, we know little of the immune status of patients in the immediate postinjury phase and how this might influence patient outcomes. The objective of this study was therefore to comprehensively assess the ultra-early, within 1-hour, immune response to trauma and perform an exploratory analysis of its relationship with the development of multiple organ dysfunction syndrome (MODS). METHODS AND FINDINGS: The immune and inflammatory response to trauma was analysed in 89 adult trauma patients (mean age 41 years, range 18-90 years, 75 males) with a mean injury severity score (ISS) of 24 (range 9-66), from whom blood samples were acquired within 1 hour of injury (mean time to sample 42 minutes, range 17-60 minutes). Within minutes of trauma, a comprehensive leukocytosis, elevated serum pro- and anti-inflammatory cytokines, and evidence of innate cell activation that included neutrophil extracellular trap generation and elevated surface expression of toll-like receptor 2 and CD11b on monocytes and neutrophils, respectively, were observed. Features consistent with immune compromise were also detected, notably elevated numbers of immune suppressive CD16BRIGHT CD62LDIM neutrophils (82.07 x 106/l ± 18.94 control versus 1,092 x 106/l ± 165 trauma, p < 0.0005) and CD14+HLA-DRlow/- monocytes (34.96 x 106/l ± 4.48 control versus 95.72 x 106/l ± 8.0 trauma, p < 0.05) and reduced leukocyte cytokine secretion in response to lipopolysaccharide stimulation. Exploratory analysis via binary logistic regression found a potential association between absolute natural killer T (NKT) cell numbers and the subsequent development of MODS. Study limitations include the relatively small sample size and the absence of data relating to adaptive immune cell function. CONCLUSIONS: Our study highlighted the dynamic and complex nature of the immune response to trauma, with immune alterations consistent with both activation and suppression evident within 1 hour of injury. The relationship of these changes, especially in NKT cell numbers, to patient outcomes such as MODS warrants further investigation.

[Dynamics of immunological features of oral liquid in children with chronic catarrhal gingivitis living on polluted territories with fluoride and iodine deficiency]. / Dinamika immunologicheskikh pokazatelei rotovoi zhidkosti u detei s khronicheskim kataral'nym gingivitom, prozhivaiushchikh na zagriaznennykh territoriiakh s ftor- i ioddefitsitom.

The aim of the study was to assess the state of oral liquid (OL) immunity in children with chronic catarrhal gingivitis (CCG) living in adverse environmental conditions. 120 children aged 7-15 residing in ecologically unfavorable areas of Lviv region were examined, while 75 children living in 'relatively clean' region were involved in the control group. Chronic catarrhal gingivitis was diagnosed according to Danilevskiy classification (1994). The level of cytokines in oral liquid of 7-years-old children living in ecologically polluted region (EPR), was (198.19±4.11)·106/l, which was 1.4 times more than in the conditionally clean region (CCR): (141.09±4.10)·106/l (p<0.01). Analysis of cytokine profile in 7-years-old from EPR showed increased levels of IL-6 proinflammatory cytokines by 11.22% (13.78±0.38 pg/ml vs 12.39±0.50 pg/ml in controls, p<0.05) and the decrease of IL-4 anti-inflammatory cytokine by 26.9% (7.12±0.62 pg/ml vs 9.74±0.58 pg/ml, p<0.01). In 12 years-old from EPR quantity of leukocytes in OL was 1.3 times higher than in controls ((246.81±4.16)·106/l vs (190.02±4.11)·106/l, p<0.01), the increase of the IL-6 content of 27.1% (p<0.01) and reduce of the IL-4 of 21.5% (p<0.05) compared to controls was also seen. At the age of 15 further increase of leucocytes in children from EPR was revealed: (297.53±4.15)·106/l, which was 1.2 times higher than in controls (p<0.01). Changes of cytokine profile in this age group were characterized by increased content of IL-6 of 26.41% (p<0.05) and IL-4 drop of 28.53% (p>0.05). Thus the age-dependent trend for the increase of leukocytes count in OL and pro-inflammatory cytokine IL-6 with the decrease of anti-inflammatory cytokine IL-4 is noted in children with CCG living in EPR.

Resistance exercise attenuates skeletal muscle oxidative stress, systemic pro-inflammatory state, and cachexia in Walker-256 tumor-bearing rats.

The aim of this study was to investigate the effects of resistance exercise training (RET) on oxidative stress, systemic inflammatory markers, and muscle wasting in Walker-256 tumor-bearing rats. Male (Wistar) rats were divided into 4 groups: sedentary controls (n = 9), tumor-bearing (n = 9), exercised (n = 9), and tumor-bearing exercised (n = 10). Exercised and tumor-bearing exercised rats were exposed to resistance exercise of climbing a ladder apparatus with weights tied to their tails for 6 weeks. The physical activity of control and tumor-bearing rats was confined to the space of the cage. After this period, tumor-bearing and tumor-bearing exercised animals were inoculated subcutaneously with Walker-256 tumor cells (11.0 × 107 cells in 0.5 mL of phosphate-buffered saline) while control and exercised rats were injected with vehicle. Following inoculation, rats maintained resistance exercise training (exercised and tumor-bearing exercised) or sedentary behavior (control and tumor-bearing) for 12 more days, after which they were euthanized. Results showed muscle wasting in the tumor-bearing group, with body weight loss, increased systemic leukocytes, and inflammatory interleukins as well as muscular oxidative stress and reduced mTOR signaling. In contrast, RET in the tumor-bearing exercised group was able to mitigate the reduced body weight and muscle wasting with the attenuation of muscle oxidative stress and systemic inflammatory markers. RET also prevented loss of muscle strength associated with tumor development. RET, however, did not prevent the muscle proteolysis signaling via FBXO32 gene messenger RNA expression in the tumor-bearing group. In conclusion, RET performed prior tumor implantation prevents cachexia development by attenuating tumor-induced systemic pro-inflammatory condition with muscle oxidative stress and muscle damage.

Postoperative leukocyte changes in facial fracture patients: a randomized prospective study with short-term dexamethasone.

PURPOSE: We investigated leukocyte changes in facial fracture patients undergoing surgery. Of specific interest was the effect of perioperative dexamethasone on leukocyte changes. METHODS: Facial fracture patients were randomized to receive perioperatively a total dose of 30 mg of dexamethasone, whereas patients in the control group received no glucocorticoid. All patients received antibiotics until postoperative days 7-10. Leukocyte count was measured on postoperative days 1 and 2. Clinical infections were observed during the follow-up. RESULTS: A total of 110 adult patients were included in the study. Postoperative leukocytosis was found in 91.2% of patients receiving dexamethasone and in 67.9% of controls. Dexamethasone was associated strongly with leukocyte rise (p < 0.001) on both postoperative days. Transoral surgery and younger age (≤40 years) showed significant associations with leukocytosis on the first postoperative day (p = 0.002). In regression analyses, dexamethasone associated with leukocytosis most significantly (p < 0.001). No association was found with infections. CONCLUSIONS: Dexamethasone use was the most significant predictor of leukocyte rise. As a drug response, perioperative dexamethasone caused sixfold postoperative leukocytosis. High-dose dexamethasone-induced leukocytosis may confuse the clinical decision-making especially in assessment of early infections.

Proinflammatory Effects of Hypoglycemia in Humans With or Without Diabetes.

Severe hypoglycemic events have been associated with increased cardiovascular mortality in patients with diabetes, which may be explained by hypoglycemia-induced inflammation. We used ex vivo stimulations of peripheral blood mononuclear cells (PBMCs) and monocytes obtained during hyperinsulinemic-euglycemic (5.0 mmol/L)-hypoglycemic (2.6 mmol/L) clamps in 11 healthy participants, 10 patients with type 1 diabetes and normal awareness of hypoglycemia (NAH), and 10 patients with type 1 diabetes and impaired awareness (IAH) to test whether the composition and inflammatory function of immune cells adapt to a more proinflammatory state after hypoglycemia. Hypoglycemia increased leukocyte numbers in healthy control participants and patients with NAH but not in patients with IAH. Leukocytosis strongly correlated with the adrenaline response to hypoglycemia. Ex vivo, PBMCs and monocytes displayed a more robust cytokine response to microbial stimulation after hypoglycemia compared with euglycemia, although it was less pronounced in patients with IAH. Of note, hypoglycemia increased the expression of markers of demargination and inflammation in PBMCs. We conclude that hypoglycemia promotes mobilization of specific leukocyte subsets from the marginal pool and induces proinflammatory functional changes in immune cells. Inflammatory responses were less pronounced in IAH, indicating that counterregulatory hormone responses are key modulators of hypoglycemia-induced proinflammatory effects. Hypoglycemia-induced proinflammatory changes may promote a sustained inflammatory state.

Biomarkers of chemotaxis and inflammation in cerebrospinal fluid and serum in individuals with HIV-1 subtype C versus B.

A defective chemokine motif in the HIV-1 Tat protein has been hypothesized to alter central nervous system cellular trafficking and inflammation, rendering HIV-1 subtype C less neuropathogenic than B. To evaluate this hypothesis, we compared biomarkers of cellular chemotaxis and inflammation in cerebrospinal fluid (CSF) and serum in individuals infected with HIV-1 subtypes B (n = 27) and C (n = 25) from Curitiba, Brazil. None had opportunistic infections. Chemokines (MCP-1, MIP-1α, MIP-1ß, RANTES, IP-10) and cytokines (TNF-α, IFN-γ, IL-1ß, IL-2, IL-4, IL-6, IL-7, IL-10) were measured using the multiplex bead suspension array immunoassays or ELISA HD. CSF and serum biomarker concentrations were compared between subtype B and C groups and HIV-positive and HIV-negative subjects (N = 19) using an independent group t test (unadjusted analysis) and linear regression (adjusted analysis), controlling for nadir CD4 and CSF and plasma HIV RNA suppression. CSF levels of cytokines and chemokines were significantly (p < 0.05) elevated in HIV-positive versus HIV-negative participants for 7/13 biomarkers measured, but levels did not differ for subtypes B and C. Serum levels were significantly elevated for 4/13 markers, with no significant differences between subtypes B and C. Although pleocytosis was much more frequent in HIV-positive than in HIV-negative individuals (27 vs. 0 %), subtypes B and C did not differ (32 and 22 %; p = 0.23). We did not find molecular evidence to support the hypothesis that intrathecal chemotaxis and inflammation is less in HIV-1 subtype C than in subtype B. Biomarker changes in CSF were more robust than in serum, suggesting compartmentalization of the immunological response to HIV.