γ' fibrinogen levels as a biomarker of COVID-19 respiratory disease severity.

Coronavirus disease 2019 (COVID-19) is characterized by a pro-inflammatory state associated with organ failure, thrombosis, and death. We investigated a novel inflammatory biomarker, γ' fibrinogen (GPF), in 103 hospitalized patients with COVID-19 and 19 healthy controls. We found significant associations between GPF levels and the severity of COVID-19 as judged by blood oxygen saturation (SpO2). The mean level of GPF in the patients with COVID-19 was significantly higher than in controls (69.8 (95 % CI 64.8-74.8) mg/dL compared with 36.9 (95 % CI 31.4-42.4) mg/dL, p < 0.0001), whereas C-reactive protein (CRP), lactate dehydrogenase (LDH), and total fibrinogen levels were not significantly different between groups. Mean GPF levels were significantly highest in patients with severe COVID-19 (SpO2 ≤ 93 %, GPF 75.2 (95 % CI 68.7-81.8) mg/dL), compared to mild/moderate COVID-19 (SpO2 > 93 %, GPF 62.5 (95 % CI 55.0-70.0) mg/dL, p = 0.01, AUC of 0.68, 95 % CI 0.57-0.78; Youden's index cutpoint 62.9 mg/dL, sensitivity 0.64, specificity 0.63). In contrast, CRP, interleukin-6, ferritin, LDH, D-dimers, and total fibrinogen had weaker associations with COVID-19 disease severity (all ROC curves with lower AUCs). Thus, GPF may be a useful inflammatory marker of COVID-19 respiratory disease severity.

Gamma' fibrinogen levels as a biomarker of COVID-19 respiratory disease severity.

Coronavirus disease 2019 (COVID-19) is characterized by a pro-inflammatory state associated with organ failure, thrombosis, and death. We investigated a novel inflammatory biomarker, γ' fibrinogen (GPF), in 103 hospitalized patients with COVID-19 and 19 healthy controls. We found significant associations between GPF levels and the severity of COVID-19 as judged by blood oxygen saturation (SpO 2 ). The mean level of GPF in the patients with COVID-19 was significantly higher than in controls (69.8 (95% CI 64.8-74.8) mg/dL compared with 36.9 (95% CI 31.4-42.4) mg/dL, p < 0.0001), whereas C-reactive protein (CRP), lactate dehydrogenase (LDH), and total fibrinogen levels were not significantly different between groups. Mean GPF levels were significantly highest in patients with severe COVID-19 (SpO 2 ≤ 93%, GPF 75.2 (95% CI 68.7-81.8) mg/dL), compared to mild/moderate COVID-19 (SpO 2 > 93%, GPF 62.5 (95% CI 55.0-70.0) mg/dL, p = 0.01, AUC of 0.68, 95% CI 0.57-0.78; Youden's index cutpoint 62.9 mg/dL, sensitivity 0.64, specificity 0.63). In contrast, CRP, interleukin-6, ferritin, LDH, D-dimers, and total fibrinogen had weaker associations with COVID-19 disease severity (all ROC curves with lower AUCs). Thus, GPF may be a useful inflammatory marker of COVID-19 respiratory disease severity.

Effect of Bacillus subtilis PB6, a natural probiotic on colon mucosal inflammation and plasma cytokines levels in inflammatory bowel disease.

The pathophysiology of inflammatory bowel disease (IBD) involves the production of diverse lipid mediators, namely eicosanoid, lysophospholipids, and platelet-activating factor, in which phospholipase A2 (PLA2) is the key enzyme. Thus, it has been postulated that control of lipid mediators production by inhibition of PLA2 would be useful for the treatment of IBD. This hypothesis has been tested in the present study by examining the therapeutic effect of a novel natural probitic Bacillus subtilis PB6 (ATCC- PTA 6737). B. subtilis PB6 is found to secrete surfactins (cyclic lipopeptides) which have anti-bacterial potential. These surfactins inhibit PLA2, a rate-limiting enzyme involved in the arachidonic acid associated inflammatory pathway and could downregulate the inflammatory response by regulating the eicosanoid and cytokine pathways. With this concept, an experimental animal trial has been conducted in a rat model of 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)-induced colitis. The oral administration of PB6 suppresses the colitis as measured by mortality rate, changes in the weight gain, colon morphology and the levels of plasma cytokines. The animals treated orally with PB6 at 1.5 x 10(8) CFU/kg thrice daily from day 4 to 10 significantly improve gross pathology of the colon and regain the colon weight to normal (p < 0.05), compared to TNBS-induced positive control. The plasma levels of pro-inflammatory cytokines (TNF-alpha, 1L-1beta, IL-6 and IFN-gamma) are also significantly lowered (p < 0.05) and anti-inflammatory cytokine (IL-I0 and TGF-beta) significantly (p < 0.05) increased after the oral administration of PB6 on day 11. The present study supports the concept that PB6 inhibits PLA2 by the secreting surfactins. In a clinical investigation, it is found to be well tolerated by all the healthy volunteers.

Isolation of genomic DNA using magnetic nanoparticles as a solid-phase support.

In recent years, techniques employing magnetizable solid-phase supports (MSPS) have found application in numerous biological fields. This magnetic separation procedure offers several advantages in terms of subjecting the analyte to very little mechanical stress compared to other methods. Secondly, these methods are non-laborious, cheap, and often highly scalable. The current paper details a genomic DNA isolation method optimized in our laboratory using magnetic nanoparticles as a solid-phase support. The quality and yields of the isolated DNA from all the samples using magnetic nanoparticles were higher or equivalent to the traditional DNA extraction procedures. Additionally, the magnetic method takes less than 15 min to extract polymerase chain reaction (PCR) ready genomic DNA as against several hours taken by traditional phenol-chloroform extraction protocols. Moreover, the isolated DNA was found to be compatible in PCR amplification and restriction endonuclease digestion. The developed procedure is quick, inexpensive, robust, and it does not require the use of organic solvents or sophisticated instruments, which makes it more amenable to automation and miniaturization.

Activity and stability of alkaline phosphatase (ALP) immobilized onto magnetic nanoparticles (Fe3O4).

Direct binding of alkaline phosphatase (ALP) on magnetic nanoparticles (Fe(3)O(4)) in the presence of carbodiimide (CDI) using two different methods is described. The activity and stability of immobilized ALP with both shaking and sonication method were compared. The results indicated the ALP binding efficiency to be in the range of 80-100% with both the immobilization techniques. The activities retained were in the range of 20-38% with shaking method and 30-43% with sonication method, respectively. The activities of the immobilized ALP preparations were found to be stable compared to the free (unbound) ALP for at least 16-week storage period. Moreover, ALP immobilized on magnetic nanoparticles was successfully used for dephosphorylation of plasmid DNA before it was used for ligation reaction. The use of immobilized ALP for plasmid dephosphorylation allows easy manipulation, reduces the procedural time, and also avoids exposure of reaction mixture to high temperature.

Enhancement in recovery of drugs with high protein binding efficiency from human plasma using magnetic nanoparticles.

In this paper, we propose an alternate method for bioanalytical extraction of drugs from human plasma samples using bare magnetic nanoparticles. The magnetic nanoparticles (MNPs) were used for deproteination of biological samples that further assist in extraction of plasma bound drugs for bioanalytical studies. The method uses basic solvents (ethanol, methanol, etc.) rather than the expensive and toxic solvents. The MNPs provide several advantages like avoiding the use of centrifuge machine, and making extraction time effective. The average time involved for the sample preparation is around 30-40min. The developed method was examined for seven different drugs having moderate (40-70%) to high (>80%) plasma protein binding efficiency. The present study focuses on the principle of magnetic nanoparticle based extraction of drug that binds with the plasma protein. In calcitriol (protein binding efficiency >99%), it was observed that the drug extraction efficiency could be enhanced by 16% using the present method. However, we assume that still there is a scope for improving the extraction efficiency by optimizing proper solvent for the specific drug. The use of magnetic nanoparticles makes the extraction cost effective and quick with improved efficiency.

Is the polymorphic microsatellite repeat of the dopamine beta-hydroxylase gene associated with biochemical variability of the catecholamine pathway in schizophrenia?

Six allelic fragments were typed by a polymerase chain reaction process with a pair of primers specific for a sequence containing the polymorphic (GT)n repeat, a microsatellite repeat, in the human dopamine beta-hydroxylase (DBH) gene. Their frequencies in unrelated patients with schizophrenia were 0.003 (A1), 0.114 (A2), 0.343 (A3), 0.526 (A4), 0.006 (A5), and 0.009 (A6), and in unrelated control subjects, 0.012 (A1), 0.086 (A2), 0.309 (A3), 0.574 (A4), 0.006 (A5), and 0.012 (A6). Kruskal-Wallis analysis revealed significant differences among the three groups, the drug-free and drug-treated patients, and the control subjects, in serum DBH activity of the subjects whose genotype was A2/A3 (H = 6.0, p < .05) or A3/A3 (H = 9.8, p < .01), in serum homovanillic acid concentration of those whose genotype was A3/A4 (H = 7.7, p < .025), and in serum tyrosine concentration of those whose genotype was A4/A4 (H = 8.3, p < .02). Mann-Whitney U test showed that in the subjects carrying the A3/A4 genotype, serum noradrenaline concentration of drug-treated patients was significantly higher than that of control subjects (N = 58, p < .02). These results suggest that genotypic polymorphism of the human DBH is likely to be associated with biochemical variability of the catecholamine pathway in schizophrenia.

Free radical scavenging systems and the effect of peroxide damage in aged human skin fibroblasts.

One prominent theory of aging postulates an accumulation of cell damage resulting from nonenzymatic chemical reactions between important cellular components and free radicals. Fibroblast lines derived from skin biopsies of psychiatric patients ranging in age from 22 to 70 were evaluated soon after adaptation to culture. No significant correlation was found between donor age and the detoxification enzyme activities of superoxide dismutase (SOD) or aryl hydrocarbon hydroxylase (AHH) or susceptibility to damage by oxygen metabolites as measured by cell viability or lactate dehydrogenase (LDH) leakage.

Studies on concentrations of NA and HVA and activity of DBH in the serum from schizophrenic patients, first-degree relatives and normal subjects.

The serum noradrenaline (NA), homovanillic acid (HVA) and dopamine beta-hydroxylase (DBH) have been examined in neuroleptic-free and -treated patients, healthy first-degree relatives of the patients and normal subjects. Analysis of variance (ANOVA) revealed significant differences in the concentrations of serum NA(F = 2.91, p < 0.05) and HVA (F = 3.58, p < 0.05), and in the activity of serum DBH (F = 2.77, p < 0.05) among the four groups. The serum NA was significantly higher in neuroleptic-free patients (475 +/- 220 pg/ml, n = 18), than in healthy first-degree relatives (343 +/- 189 pg/ml, n = 37, p < 0.05) or in normal subjects (354 +/- 111 pg/ml, n = 17, p < 0.05), and it also was significantly higher in neuroleptic-treated patients (442 +/- 223 pg/ml, n = 58) than in healthy first-degree relatives (p < 0.05) or in normal subjects (p < 0.05). There was a trend towards high serum HVA in neuroleptic-free patients (11.3 +/- 6.3 ng/ml, n = 17) compared with the other three groups. The serum DBH activity was high in neuroleptic-free patients (31.2 +/- 15.6 nmol/min/ml, n = 17), and significantly in comparison with those treated with neuroleptic drugs (21.6 +/- 10.9 nmol/min/ml, n = 56, p < 0.05). There was a significant negative correlation between HVA concentration and DBH activity in the serum from neuroleptic-free patients (r = -0.64, n = 16, p < 0.01), and there appeared to be three subgroups with alterations of serum DBH activity and HVA concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

A study of the relationship between the DBH activity in serum and a MspI polymorphic site in intron 9 of the human DBH gene in schizophrenia.

Intron 9 of the human dopamine beta-hydroxylase (DBH) gene was amplified using a long PCR procedure in unrelated patients with schizophrenia and unrelated control subjects. MspI digestion of the PCR fragments showed a two allele polymorphism. A1 and A2 Kruskal-Wallis analysis revealed a significant difference in serum DBH activity among the three groups carrying the A1/A2 genotype, the drug-free and drug treated patients, and the control subjects (H = 12.2, df = 2, p < 0.005), and also a significant difference among the three subgroups of drug-treated patients carrying the genotype of A1/A1, A2/A2 or A1/A2 (H = 10.4, df = 2, p < 0.01). The present results suggest that the MspI polymorphic site in intron 9 of the human DBH gene may be associated with alterations of DBH activity in schizophrenia and with the influence of neuroleptic drugs on the DBH activity as well.

Role of polyamines in the membrane pathology of schizophrenia. A study using fibroblasts from schizophrenic patients and normal controls.

The polyamines putrescine, spermine, and spermidine play a major role in the regulation of cell growth and differentiation, metabolic pathways, and on cell membrane functions in mammalian systems. It has recently been suggested that polyamines may be involved in the pathophysiology of schizophrenia. Moreover, several reports suggest that schizophrenia may be associated with a generalized cell membrane abnormality. In view of these findings, we measured polyamine levels in cultured skin fibroblasts from schizophrenic patients and normal control subjects. These was a significant increase in the levels of spermidine and in total polyamines in fibroblasts and spermine in the culture medium from schizophrenic patients. This preliminary report suggests that polyamines may play an important role in the membrane abnormalities that have been reported in schizophrenic patients.

A study of enzymes involved in catecholamine metabolism in parents of patients with schizophrenia.

The concentrations of serum homovanillic acid (HVA), norepinephrine (NE), tyrosine (Tyr), phenylalanine (Phe) and tryptophan (Trp), and the activities of serum dopamine-beta-hydroxylase (DBH), platelet monoamine oxidase (MAO), and erythrocyte catechol-O-methyl transferase (COMT) were measured in 68 healthy parents who had schizophrenic offspring. The results show a significant correlation between the parents of schizophrenic patients in serum HVA (r=0.38, n=34, p<0.05), NE (r=0.40, n=33, p<0.02), Phe (r=0.44, n=34, p<0.0l), Tyr (r=0.43, n = 34, p <0.02) and DBH activity (r=0.51, n = 30, p <0.005), but do not show a significant correlation in erythrocyte COMT (r=0.01, n=27), platelet MAO (r=0.04, n=23) or serum Trp (r=0.10, n=34). There were no significant correlations in these measurements between randomly matched parents. The present study suggests that both parental sides of schizophrenic patients are likely to have similar alleles associated with the catecholamine pathway, and their ill offspring may possess a double dose of the schizophrenogenic alleles.

Comparisons of morphology and growth rate between cells from skin biopsies of schizophrenic patients and those from normal individuals.

Skin biopsies have been used to establish cell cultures from ten schizophrenic individuals and ten controls. Studies of the time of outgrowth of different cell types from the tissue fragments and the subsequent growth rates and morphology showed no difference between the primary cultures from the schizophrenic patients and controls. The primary cultures were trypsinized and grown for three passages before storing in liquid nitrogen. No difference between cell strains established from schizophrenic skin donors and cell strains from healthy controls was noted in this phase of growth. Aliquots of cells were thawed out from liquid nitrogen, grown for one passage and then the cell aggregation rate was tested. No difference was found between cells from schizophrenic skin donors and cells from controls. Differences between the two cell strains established from certain individuals did occur, suggesting that morphology and growth rates should be noted before cell strains are used for further studies.

Low concentrations of serum tyrosine in neuroleptic-free schizophrenics with an early onset.

The concentrations of serum tyrosine, phenylalanine and tryptophan have been determined in 23 male neuroleptic-free patients with schizophrenia and 28 male healthy control subjects. Tyrosine was significantly lower in neuroleptic-free patients with an early onset (starting before adulthood) than in healthy control subjects (p < 0.05), and the ratio of tyrosine to phenylalanine was significantly lower in neuroleptic-free patients with an early onset than in those with a late onset (starting at adulthood). No significant differences in these three amino acids were found between the patients with a late onset and healthy control subjects. The present findings suggest that there may be a disturbance of balance between tyrosine and phenylalanine in early-onset patients with schizophrenia.

Two double-blind placebo-controlled pilot studies of eicosapentaenoic acid in the treatment of schizophrenia.

Evidence that the metabolism of phospholipids and polyunsaturated fatty acids (PUFA) is abnormal in schizophrenia provided the rationale for intervention studies using PUFA supplementation. An initial open label study indicating efficacy for n-3 PUFA in schizophrenia led to two small double-blind pilot studies. The first study was designed to distinguish between the possible effects of two different n-3 PUFA: eicosapentaenoic acid (EPA) and docohexaenoic acid (DHA). Forty-five schizophrenic patients on stable antipsychotic medication who were still symptomatic were treated with either EPA, DHA or placebo for 3 months. Improvement on EPA measured by the Positive and Negative Syndrome Scale (PANSS) was statistically superior to both DHA and placebo using changes in percentage scores on the total PANSS. EPA was significantly superior to DHA for positive symptoms using ANOVA for repeated measures. In the second placebo-controlled study, EPA was used as a sole treatment, though the use of antipsychotic drugs was still permitted if this was clinically imperative. By the end of the study, all 12 patients on placebo, but only eight out of 14 patients on EPA, were taking antipsychotic drugs. Despite this, patients taking EPA had significantly lower scores on the PANSS rating scale by the end of the study. It is concluded that EPA may represent a new treatment approach to schizophrenia, and this requires investigation by large-scale placebo-controlled trials.

Association of polymorphic VNTR region in the first intron of the human TH gene with disturbances of the catecholamine pathway in schizophrenia.

In the present study, five allelic fragments were typed by a polymerase chain reaction (PCR) process with a pair of primers specific for the tetranucleotide (TCAT) repeat sequence in the first intron of the human tyrosine hydroxylase (TH) gene and their sizes (bp) were 114 (A), 118 (B), 122 (C), 126 (D) and 130 (E), respectively. The AE genotypic frequency was found to be significantly higher in unrelated patients with schizophrenia than in unrelated control subjects (chi 2 = 4.18, p < 0.05). ANOVA revealed a significant difference between the three groups (neuroleptic-free patients possessing or not possessing the AE genotype, and unrelated control subjects) in the concentration of serum noradrenaline (F = 4.96, df = 2.79, p < 0.01), but no significant differences were found between the three groups in the concentrations of serum homovanillic acid, phenylalanine and tyrosine. These results suggest that the polymorphic intron 1 of the human TH gene may be associated with disturbances of the catecholamine pathway in schizophrenia.

TaqI polymorphic sites at the human dopamine beta-hydroxylase gene possibly associated with biochemical alterations of the catecholamine pathway in schizophrenia.

Two parts of the dopamine beta-hydroxylase (DBH) gene, one a 7.5-kb single copy fragment (F1) spanning the 5'-flanking region to exon 3 and the second a 9.0-kb single copy fragment (F2) spanning exon 3 to exon 7, were amplified by a long PCR procedure in 161 unrelated patients with schizophrenia and 67 unrelated control subjects. The PCR products were completely digested with the restriction enzyme TaqI. These subjects were classified into genetic subgroups according to the TaqI restriction fragment length polymorphisms (RFLPs) for the human DBH gene, and the association of the TaqI RFLPs with biochemical alterations of the catecholamine pathway in schizophrenia was then examined. The frequencies of the two TaqI polymorphic sites did not show significant differences between the patients and control subjects, but the TaqI RFLPs were found to be associated with biochemical alterations of the catecholamine pathway in schizophrenia.

Association of the Ban I dimorphic site at the human cytosolic phospholipase A2 gene with schizophrenia.

There is evidence of increased phospholipid breakdown in cell membranes of patients suffering from schizophrenia. This may be related to increased levels of the enzyme cytosolic phospholipase A2 (cPLA2) which have been reported in schizophrenic subjects. We have identified a Ban I dimorphic site on the first intron of the cPLA2 gene. Schizophrenic subjects were found to have a significant excess of the A2/A2 homozygote relative to healthy control subjects. Genetically determined alterations in phospholipase activity may thus underlie the reported abnormalities of phospholipid metabolism in schizophrenia.

Plasma homovanillic acid in untreated schizophrenia--relationship with symptomatology and sex.

Plasma homovanillic acid (pHVA) concentrations are considered to reflect, in part, central dopamine metabolism and thus may be of value in assessing the role of dopamine neurotransmission in schizophrenia. Furthermore, some recent studies have suggested a relationship of pHVA with symptomatology. We have undertaken a study of pHVA in a large cohort of unmedicated DSM-IV schizophrenic patients in order to assess the relationship of pHVA to various clinical parameters. pHVA in 58 drug-free patients (10.11+/-0.52 ng/ml) was significantly elevated in comparison with 62 matched control subjects (8.77+/-0.39 ng/ml). pHVA was found to be higher in patients with a more negative syndrome. No significant correlation of pHVA with overall SAPS or SANS scores was apparent in the patients although, within the SANS subscales, a significant relationship to anhedonia-asociality was apparent. Interestingly, the male drug-free patients showed a correlation of pHVA with negative symptoms defined by SANS and several SANS subscales, while females showed no significant relationship with any SANS subscales. The results may suggest that an increased dopaminergic turnover is apparent in (male) schizophrenic patients with predominantly negative symptoms, providing some support for reports that this change in neuronal activity may be related to the neuropathological abnormalities seen in the disease, which may themselves differ between males and females. Such neuronal deficits of developmental or degenerative origin may thus result in an elevation/disinhibition of central dopamine metabolism in schizophrenia.