Complement Heat Tolerance as a Marker of Protein Fragility and Its Clinical Significance

Abstract

This study aimed to establish a complement tolerance test (CTT) as a marker of protein fragility and discuss its clinical significance. Total complement activity (TCA) of serum was measured using a self-hemolysis colorimetric method. Human O-erythrocytes and rabbit anti-human O-erythrocyte antibodies were used to replace sheep erythrocytes and the corresponding hemolysin for the hemolysis test, respectively. The antigen-antibody specific binding activated the classical pathway of complement, generating a membrane attack complex, and the red blood cells rupture. A CTT was established to measure complement heat tolerance according to the sensitivity of complement proteins to temperature, which was calculated according to differences in TCA at different temperatures. The smaller the CTT the stronger the complement resistance to heat. The method was applied to the detection of diabetic patients and healthy controls. The mean value of CTT (mean) = 0.063 ± 0.003 with a coefficient of variation of 4.8% for the same specimen tested for complementary thermal resistance on 5 consecutive days, which is a good stability of the assay. Application of CTT on samples from patients with different ages revealed significantly higher mean CTT values for elderly patients (≥60-years old) relative to those for younger patients (20–40-years old) (p < 0.05). In addition, the mean CTT values for diabetic patients were significantly higher than those for healthy patients (p < 0.001). We successfully established a method that uses complement thermal resistance as a marker of protein fragility, with the results demonstrating the ability of the CTT identify age- and disease-related variations in patient samples and its potential efficacy for clinical application.

Introduction

The gradual inability of cells and organisms to maintain protein homeostasis with age and the reduced ability of proteins to resist external perturbations lead to a decline in the overall health of the organism, inadequate response to stress, and a shortened healthy lifespan.¹ Maintenance of cellular protein homeostasis is a key process that ensures longevity, with primary factors involved in this process, including chaperones and two protein-hydrolysis systems: the ubiquitin–proteasome and the lysosome–autophagy system.² Notably, both autophagy and proteasome activity decrease with age,³ which is accompanied by enhanced protein damage due to oxidation that ultimately interferes with normal chaperone function. These actions result in imbalanced protein homeostasis⁴ and increased vulnerability of the proteome to stress in the aging population.³

In addition, heat treatment, which induces protein denaturation and aggregation, affects protein spatial structure and function due to conformational changes. A previous study identified caloric restriction as an effective intervention to combat aging and improve stress resistance in cultured cells based on improved protein stability,⁴ These findings suggest that protein vulnerability to external stimuli might be age-related.

However, the change of protein is also related to the redox state of the body, diseases, and other factors. Compared with the younger group, it was found that the total antioxidant capacity in the older than 75-year age group decreased significantly, along with a decrease in the mass of antioxidant substances and average activity levels in human serum.⁵ In contrast to the results in serum, more antioxidants were detected in the urine of the elderly in the healthy population. In addition, more antioxidants were found in urine of tumor patients with same age range.⁶ The antioxidant capacity of serum and urine was significantly lower in diabetic patients compared to healthy individuals.⁷

Antibody activity was determined by a variety of immunological assays, including precipitation reactions, agglutination reactions, and enzyme immunoassays, to quantitatively assess the effect of oxidants on antigen-antibody binding in vitro. A certain concentration of oxidants significantly inhibited the activity of antibodies, but had little effect on the total antioxidant capacity of serum.⁸ In addition, a certain concentration of ammonium ferrous sulfate could significantly inhibit antibody, enzyme, DNA, and diluted serum. Certain concentrations of ascorbic acid resulted in significant inhibition of antibody. The reduction in antibody activity induced by oxidizing agents was partially repaired by a reductant.⁹

Aging attenuates the efficiency of bodily functions. Biomarkers of this vulnerability have been identified based on the relationship between aging and age-related diseases or lifespan. These include markers of inflammation (e.g., interleukin-6 and C-reactive protein), the immune response (e.g., white blood cell count), and epigenetic changes (e.g., DNA methylation), as well as clinical markers, such as albumin and fibrinogen levels.¹⁰ Because protein destabilization during aging is related to the pathogenesis of numerous diseases, measurement of protein fragility might enable identification and prediction of those at risk of age-related diseases to optimize intervention and treatment.

The complement system comprises heat-resistant proteins present in normal human and animal serum and plays an important role in host defense against bacteria, the interface between innate and adaptive immunity, and removal of immune complexes and apoptotic cells.¹¹ The three main complement-activation pathways (classical, lectin, and alternative) promote the formation of a membrane-attacking complex (MAC) that regulates phagocytosis or lysis of pathogenic targets. Changes in the activity/function of any component of this system adversely effect the complement system, thereby altering its ability to regulate the destruction of invading microorganisms and prevent excessive activation of autologous cells and tissues.¹²

The activation of complement is a cascade of enzymatic reactions and is very sensitive to temperature. Affected by heat treatment, heat intolerance of any of the proteins of this system leads to changes in the total complement activity (TCA). Thus the fragility of the protein can be reflected by the complement tolerance test (CTT). There are neither detailed studies on complement heat tolerance nor are there any reports using it as a marker of protein fragility. In this study, the change in TCA during heating to reflect protein fragility, which may be associated with the onset of certain diseases, is a new indicator for disease diagnosis.

Moreover, age-related alterations in protein stability can affect the complement system, which is also highly sensitive to changes in temperature.¹³ Therefore, evaluation of complement thermal resistance might reflect overall levels of protein stability according to their ability to resist external disturbance. The CTT was established to measure complement heat tolerance according to the sensitivity of complement proteins to temperature, which was calculated according to differences in the TCA at different temperatures.

The main methods used for the determination of TCA include the CP-CH50 method, the AP-CH50 method, the single-tube titration method, and the plate-hemolysis method. It has been established that the complement system is heat-labile and that heating the serum samples above 37℃ could denature some of the complement proteins and thus resulting in lower activity as measured by the CH50 assay. Therefore, in this study, we used a previous technique for determining complement efficacy that uses antigen–antibody complexes to assess 50% hemolytic complement activity (CH50).¹⁴ We then developed a colorimetric method to measure TCA according to the CH50 using human red blood cells (RBCs),¹⁵ which allowed evaluation of changes in complement activity according to temperature and determination of overall complement thermal resistance.

The results demonstrated the utility of the complement heat tolerance as a marker of protein fragility and its relationship with age- and disease-related changes. Meanwhile, the protein fragility in older adults may be related to a reduced capacity for oxidative stress. Oxidative stress itself is not significant when it does not cause protein alterations, but when it does cause protein damage, it affects protein fragility, and protein changes can lead to functional alterations and therefore induce disease. Finally, the complement heat resistance assays are clinically important and can be used as markers for onset of certain diseases.

Materials and Methods

All of the experiments on the subjects were conducted in accordance with the Declaration of Helsinki, the study was approved by the Institutional Ethics Committee of Dalian Medical University and informed consent was waived, as the samples were remnants following clinical use and therefore not specifically collected for this study and without risk to patients.

Establishment of a CTT to measure the complement thermal resistance

We used 2% human O-erythrocytes as a hemolysis-indicator system and the 2U hemolysin to detect the extent of hemolysis. The 2U hemolysin (anti-human O-erythrocyte antibody, trade name rabbit anti-human O-erythrocyte serum, potency 1: 100, supplied by Zhengzhou Baiji Biotechnology Co., Ltd.) was diluted in the buffer mentioned above according to potency (this concentration unit was based on the potency of hemolysin when it caused an agglutination reaction; when the potency of hemolysin was 100, we diluted it 50 times to obtain 2 U of hemolysin). We confirmed complement activity based on MAC generation and erythrocyte rupture and according to correlations between the degree of hemolysis and complement activity. Optical density (OD) values were determined at 37°C and 47°C and were used to calculate TCA, with the difference used to calculate the complement thermal resistance (Fig. 1).

FIG. 1.

Principle diagram of the complementary thermal resistance experiment. CTT, complement tolerance test; MAC, membrane-attacking complex.

The study subjects were all Chinese, and there was no genetic relationship between them. Serum was obtained by collecting venous blood from patients and healthy controls, leaving it to stand for 1 hour at 4°C, and centrifuged at 2500 rpm (1118 × g) for 5 minutes to separate the serum, which was divided into two 100-μL aliquots that were subsequently incubated at 37°C and 47°C, respectively, in a water bath for 30 minutes. Ensure that the number of samples tested is sufficiently large and that details regarding sample collection, storage, and storage duration were consistent.

Serum samples were then transferred to 1.5-mL tubes, into which the 50-μL anti-RBC antibody (1:100), 50-μL 2% human O-erythrocytes, and 800-μL PBS buffer were added (Fig. 2) for incubation at 37°C for 30 minutes. The mixture was then centrifuged at 2500 r/min for 5 minutes, and 100 μL of supernatant from each tube was added to a 96-well microtiter plate, which was used to determine the OD at 542 nm. TCA was determined by subtracting the OD values at each respective temperature.

FIG. 2.

The flowchart of experimental operation of the complement tolerance test. RBC, red blood cell.

Assessment of the complement thermal resistance assay

Serum samples were subjected to measurements of the complement thermal resistance assay for 5 consecutive days to demonstrate method reproducibility. Venous blood was collected and serum isolated, as described, followed by separation into five 200-μL aliquots, one of which was used immediately and the remaining immediately frozen at −20°C until use. Each aliquot was divided into two 100-μL samples that were used to perform the assay described in Establishment of a CTT to Measure the Complement Thermal Resistance section, which was repeated for each of the five samples on separate days.

Measurement of the complement thermal resistance using samples from different age and gender groups

Healthy adult subjects were divided into age (20–40 years old [n = 39; mean: 30.77 ± 6.98 years] and ≥60 years old [n = 49; mean: 75.20 ± 8.13 years]) and gender (25 males; mean: 30.96 ± 7.30 years; 14 females; mean: 30.43 ± 6.62 years). The CTT was performed on samples as described in Establishment of a CTT to Measure the Complement Thermal Resistance section.

Measurement of the complement thermal resistance in diabetic patients

Serum samples were collected from 42 patients (20 males, 22 females) meeting the clinical diagnostic indexes of diabetes mellitus and the World Health Organization criteria for type 2 diabetes (T2D) mellitus at the First Affiliated Hospital of Dalian Medical University (Dalian, China), with sera from 39 healthy patients (21 males, 18 females) used as controls. There were no statistical differences between the mean ages of both groups (control: 68.69 ± 6.10 years vs. diabetic: 69.10 ± 11.97 years).

Statistical analysis

Data dispersion was compared by calculating the coefficient of variation (CV). According to the data homogeneity of variance (Levene's test) and normal distribution (Kolmogorov-Smirnov test), an independent sample t test was used to determine differences between two groups of samples. All analyses were carried out using SPSS statistical analysis software (SPSS, Inc., Chicago, IL). p-Values <0.05 (two-tailed) were considered significant.

Results

CTT stability

Table 1 shows the results of measuring the complement thermal resistance on serum samples from the same healthy volunteer over 5 consecutive days. CTT results were 0.066, 0.066, 0.060, 0.060, and 0.064 (mean: 0.063 ± 0.003), with a CV of 4.8%.

Table 1.

Results of Complement Tolerance Test on Serum Samples from the Same Healthy Volunteer on 5 Consecutive Days

Of No. of days	CTT value	Mean	SD	CV
1	0.066	0.063	0.003	4.8%
2	0.066
3	0.060
4	0.060
5	0.064

CTT, complement tolerance test; CV, coefficient of variation; SD, standard deviation.

Age-related changes in CTR

Table 2 shows that the mean of the complement thermal resistance measured using the CTT differed significantly according to age (20–40 years old: 0.053 ± 0.007 vs. ≥60 years old: 0.056 ± 0.008) (p < 0.05).

Table 2.

Results of Complement Tolerance Test in Different Age Groups

Group	N	Mean	SD	K-S test (p value)	Levene's test (p value)	Student's t test
Group	N	Mean	SD	K-S test (p value)	Levene's test (p value)	t	p
20–40 years old	39	0.053	0.007	0.121	0.983	2.031	0.045
≥60 years old	49	0.056	0.008	0.096	0.983	2.031	0.045

K-S test, Kolmogorov-Smirnov test.

Gender-related differences in the complement thermal resistance

Table 3 shows no significant difference in the complement thermal resistance according to gender based on the CTT (males: 0.053 ± 0.006 vs. females: 0.052 ± 0.007) (p > 0.05).

Table 3.

Results of Complement Tolerance Test in Males and Females

Group	N	Mean	SD	K-S test (p value)	Levene's test (p value)	Student's t test
Group	N	Mean	SD	K-S test (p value)	Levene's test (p value)	t	p
Males	25	0.053	0.006	0.200	0.346	0.393	0.697
Females	14	0.052	0.007	0.200	0.346	0.393	0.697

Differences in the complement thermal resistance between diabetic patients and healthy controls

Table 4 shows a significant difference in the complement thermal resistance between diabetic patients and healthy controls (controls: 0.042 ± 0.008 vs. diabetic: 0.050 ± 0.008) (p < 0.001).

Table 4.

Results of Complement Tolerance Test in Diabetic Patients and Healthy Subjects

Group	N	Mean	SD	K-S test (p value)	Levene's test (p value)	Student's t test
Group	N	Mean	SD	K-S test (p value)	Levene's test (p value)	t	p
Healthy subjects	39	0.042	0.008	0.200	0.898	4.345	<0.001
Patients	42	0.050	0.008	0.200	0.898	4.345	<0.001

Discussion

There is some evidence to verify the reliability of our method.^16

–19 The main methods used to determine TCA include CP-CH50 method, the single-tube titration method, and the plate hemolysis method. However, the CP-CH50 method is a manual, complex, and semiquantitative analysis that requires a long preparation time. In addition, fresh sheep red blood cells are difficult to collect, transfer, and preserve, and their quality is not guaranteed. Dong et al established a method for measuring TCA based on a hemolysis system using own RBCs.¹⁵

Human RBCs were used as a hemolysis indicator system and rabbit anti-human RBC antibody instead of the traditional hemolysin (rabbit anti-sheep RBC antibody) in the new method. TCA using the new method was significantly correlated with the CH50 method and had clinical significance. In this study, the single-tube method was chosen instead of the multitube method and overcame the complicated procedure and poor stability of the CH50 method, simplified the detection of TCA.^20
–22

We established a new method for characterizing parameters of complement activity as a marker of protein fragility. As heat treatment is a physical factor, it is easy to manipulate and can affect the spatial structure and function of proteins. Heat adversely affects protein secondary structure, which alters the function of each complement component and each product of the activation process. In this study, we measured complement activity using cytolysis experiment at two temperature gradients (37°C and 47°C), as well as CTT accuracy for 5 consecutive days, finding a CV of 4.8%, which demonstrated the stability of the test. Furthermore, we confirmed the absence of significant gender-specific differences in CTT results, whereas significantly higher readings were identified in older patients relative to younger patients (old: 0.056 ± 0.008 vs. young: 0.053 ± 0.007, p < 0.05).

These findings indicated that age-related sensitivity to temperature and external stimuli alter complement protein structures. The objective of the study was to establish a CTT as a maker of protein fragility and discuss its clinical significance. While the readout of the assay is OD, the ODs are typically normalized to the OD of water (100% lysis) to calculate % lysis of each sample. This is important because the OD may change from run to run depending on the target cell preparation. This point needs to be clear throughout in the study.

In addition, we performed a case-control study using diabetic patients and age-appropriate healthy controls in the clinical setting to evaluate differences in complement protein resistance to temperature. It shows a significant difference in the complement thermal resistance between diabetic patients and healthy controls (controls: 0.042 ± 0.008 vs. diabetic: 0.050 ± 0.008). The comparison revealed significantly weaker thermal resistance in diabetic patients relative to healthy subjects (p < 0.001). For each batch of experiments, fresh 2% RBCs were prepared again, so the detection system was different. When there were different blood cells in the detection system, OD and CTT values would be slightly different, so each batch of experiments set up its own control group. It was observed that the results of different experiments were slightly different, which had no influence on the conclusion.

There is a strong link between the complement system, the activity of complement regulatory proteins, and the pathogenesis of diabetes and its complications.²³ The content of hemolytically effective molecules of some classical complement components as well as factors of the alternative activation pathway was studied by radio immunodiffusion method and other methods. Levels of C3 and C4, factors B and D were significantly elevated in the patients with insulin dependent diabetes compared with healthy donors. However, if the insulin-dependent diabetes have antigens B18, C4 levels were lower than those of the control group. In noninsulin-dependent diabetes C4, factors B and D were significantly elevated, and C5 levels were reduced. The levels of complement C3 and C5 were decreased and the content of C4 was increased in diabetic patients with diabetic nephropathy.²⁴

Furthermore, according to proteomic analysis, when comparing gestational diabetes mellitus (GDM) with controls (CG), 78 proteins were significantly changed, related to complement. In GDM, Complement C3 (C3), Complement C5 (C5), C4-B (C4B), C4b-binding protein beta chain (C4BPB), and C4b-binding protein alpha chain (C4BPA) were downregulated, while C7, C9, and F12 were upregulated.²⁵ Plasma levels of C5a, C3a, and C5b-9 were significantly increased in patients with T2D normal albuminuria.²⁶ Complement C5a induces renal injury in diabetic kidney disease by disrupting mitochondrial metabolic agility and complement C3d and C5b-9 were abundantly deposited in the choriocapillaris of eyes of patients with diabetic retinopathy.²⁷ Complement C3 was associated with foot infections in diabetic patients, and alterations in serum complement C3 may result in reduced immune responses leading to delayed wound healing and recurrent infections.²⁸

In addition, response gene to complement 32 (RGC-32) in endothelial cells might lose expression in T2D retina with diabetic retinopathy (DR) features and played a key role in DR pathogenesis.²⁹ Proteomic analysis of diabetes genetic risk scores identified complement C2 as predictor of T2D.³⁰ C1q etc. could be used as potential diagnostic or therapeutic targets for diseases such as T2D.³¹

In short, the delicate balance between complement activation and restriction in diabetes can be disrupted in different ways. Autoantibodies to glycosylated and glycosyl oxidized proteins can activate the classical pathway,^32–33 fructosamine activates the lectin pathway by increasing ligand–receptor binding³⁴ and increased arterial wall age can serve as a new epitope for mannan-binding lectin interactions.³⁵

Due to the characteristics of the enzymatic reaction cascade involving a series of proteins, changes in any component of complement will affect the cascade, causing problems in the balanced mechanism of complement. Affected by heat treatment, heat intolerance of any of the proteins of this system leads to changes in the TCA. Thus the fragility of the protein can be reflected by the CTT. This method offers pathological significance and will be useful in understanding the pathogenesis of diabetes, as well as predicting the risk of diabetes and possible interventions. Moreover, compared with healthy subjects of the same age, type 2 diabetic patients show reduced complement thermal resistance and increased protein fragility, which predispose them to age-related diseases.

Meanwhile, in reality, protein alterations are also associated with oxidation or other factors. Alterations in oxidation/reduction balance are associated with many pathological and physiological conditions, including aging, tumors, and diabetes. In healthy populations, more antioxidants were detected in the urine of older adults, while the opposite result was found in serum.⁶ In addition, it was found that the antioxidant capacity of serum and urine was significantly lower in diabetic patients compared to healthy individuals.⁷ However, any alteration in protein vulnerability due to either oxidative stress or age ultimately tends to be pathological and may be an important cause for the pathogenesis of several diseases, making this topic of great research importance. During the study, the age of the subjects in the experimental and control groups was strictly controlled to be consistent, so that the oxidative stress experienced was approximately the same, and its effects could be eliminated in the study.

This method is a blood-based assay that can be used for repeat measurements and is sensitive to age-related differences, enabling it to predict the risk of age-related diseases. Due to oxidative stress, age and other indicators all lead to functional changes through protein changes and then induce diseases. Therefore, compared with oxidative stress and other indicators, it may be more direct to reflect the degree of protein fragility by measuring the total activity of complement, which may more directly assess the redox state of some diseases and reliably reflect the disease state. Thus, it is believed that our method has the advantages of good stability, less interference, simplicity, rapidity, and sensitivity.^36

–39

Given the aging population in China, disease prevention has become a key issue, and further research on reliable indicators related to aging and geriatric diseases is of great importance to increase the health span of individuals and reduce their medical burden.

Conclusions

In summary, due to the fact that complement is not tolerant to heat and complement activation is a cascade reaction involving a series of proteins, a change in any one protein will cause a change in the reaction. In this study, a new index of protein fragility was established, and we created a CTT that uses the complement thermal resistance as a marker of protein fragility. The results indicated that the CTT was able to identify increased vulnerability of complement proteins to heat in type 2 diabetic patients, suggesting its potential clinical efficacy for predicting the risk of age-related diseases and possible interventions.

Footnotes

Authors' Contributions

L.J.H. and X.J.J. did the experimental part. L.J.H. and H.L. wrote the main article text. All authors reviewed the article.

Data Availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

No funding was received for this article.

References

Morimoto

, Cuervo

. Proteostasis and the aging proteome in health and disease. J Gerontol A Biol Sci Med Sci, 2014; 69 Suppl 1(Suppl 1):S33–S38; doi: 10.1093/gerona/glu049

Tsakiri

, Iliaki

, Höhn

, et al. Diet-derived advanced glycation end products or lipofuscin disrupts proteostasis and reduces life span in Drosophila melanogaster . Free Radic Biol Med, 2013; 65:1155–1163; doi: 10.1016/j.freeradbiomed.2013.08.186

Kaushik

, Cuervo

. Proteostasis and aging. Nat Med, 2015; 21(12):1406–1415; doi: 10.1038/nm.4001

Korovila

, Hugo

, Castro

, et al. Proteostasis, oxidative stress and aging. Redox Biol, 2017; 13:550–567; doi: 10.1016/j.redox.2017.07.008

Zhang

, Liu

. Determination of the total mass of antioxidant substances and antioxidant capacity per unit mass in serum using redox titration. Bioinorg Chem Appl, 2014; 2014:928595; doi: 10.1155/2014/928595

Zhou

, Chen

, Wang

, et al. Evaluating the risk of tumors diseases based on measurement of urinary and serumal antioxidants using the new agar diffusion methods. Oxid Med Cell Longev, 2017; 2017:6578453; doi: 10.1155/2017/6578453

Cao

, He

, Bai

, et al. Establishment of a method for measuring antioxidant capacity in urine, based on oxidation reduction potential and redox couple I2/KI. Bioinorg Chem Appl, 2016; 2016:7054049; doi: 10.1155/2016/7054049

Han

, Wang

, Xu

, et al. Quantitative assessment of the effects of oxidants on antigen-antibody binding in vitro. Oxid Med Cell Longev, 2016; 2016:1480463; doi: 10.1155/2016/1480463

Zhao

, Xu

, Liu

. Quantitative assessment of the effects of reducing agents on biological macromolecules and on the possible repair of oxidative damage. Biomed Res Int, 2018; 2018:5704016; doi: 10.1155/2018/5704016

10.

Kane

, Sinclair

. Frailty biomarkers in humans and rodents: Current approaches and future advances. Mech Ageing Dev, 2019; 180:117–128; doi: 10.1016/j.mad.2019.03.007

11.

Botto

, Kirschfink

, Macor

, et al. Complement in human diseases: Lessons from complement deficiencies. Mol Immunol, 2009; 46(14): 2774–2783 doi: 10.1016/j.molimm.2009.04.029

12.

Walport

. Complement. Second of two parts. N Engl J Med, 2001; 344(15): 1140–1144; doi: 10.1056/NEJM200104123441506

13.

Freeley

, Kemper

, Le Friec

. The “ins and outs” of complement-driven immune responses. Immunol Rev, 2016; 274(1):16–32; doi: 10.1111/imr.12472

14.

Sarma

, Ward

. The complement system. Cell Tissue Res, 2011; 343(1):227–235; doi: 10.1007/s00441-010-1034-0

15.

Dong

, Liu

. Establishment of a method for measuring total complement activity based on a hemolysis system using own red blood cells. J Immunol Methods, 2016; 430:21–27; doi: 10.1016/j.jim.2016.01.010

16.

Gutmann

, Takov

, Burnap

, et al. SARS-CoV-2 RNAemia and proteomic trajectories inform prognostication in COVID-19 patients admitted to intensive care. Nat Commun, 2021; 12(1):3406; doi: 10.1038/s41467-021-23494-1

17.

Rognes

, Pischke

, Ottestad

, et al. Increased complement activation 3 to 6h after trauma is a predictor of prolonged mechanical ventilation and multiple organ dysfunction syndrome: A prospective observational study. Mol Med, 2021; 27(1):35; doi: 10.1186/s10020-021-00286-3

18.

Nakamura

, Sueyoshi

, Miyoshi

, et al. Complement activation in patients with heat-related illnesses: Soluble CD59 Is a novel biomarker indicating severity of heat-related illnesses. Crit Care Explor, 2022; 4(4):e0678; doi: 10.1097/CCE.0000000000000678

19.

Skendros

, Mitsios

, Chrysanthopoulou

, et al. Complement and tissue factor-enriched neutrophil extracellular traps are key drivers in COVID-19 immunothrombosis. J Clin Invest, 2020; 130(11):6151–6157; doi: 10.1172/JCI141374

20.

West

, Kolev

, Kemper

. Complement and the regulation of T cell responses. Annu Rev Immunol, 2018; 36:309–338; doi: 10.1146/annurev-immunol-042617-053245

21.

Kunz

, Kemper

. Complement has brains-do intracellular complement and immunometabolism cooperate in tissue homeostasis and behavior?. Front Immunol, 2021; 12:629986; doi: 10.3389/fimmu.2021.629986

22.

, Qin

. Role of complement system in kidney transplantation: Stepping from animal models to clinical application. Front Immunol, 2022; 13:811696; doi: 10.3389/fimmu.2022.811696

23.

Ghosh

, Sahoo

, Vaidya

, et al. Role of complement and complement regulatory proteins in the complications of diabetes. Endocr Rev, 2015; 36(3):272–288; doi: 10.1210/er.2014-1099

24.

Titov

, Korvigo

, Kharitonik

, et al. Levels of various components of the classical and alternative pathways of complement activation in diabetes mellitus patients. Probl Endokrinol (Mosk), 1987; 33(2):9–13; PMID: 3474606

25.

Charlesworth

, Timmermans

, Golding

, et al. The complement system in type 1 (insulin-dependent) diabetes. Diabetologia, 1987; 30(6):372–379; doi: 10.1007/BF00292537

26.

Tan

, Ziemann

, Thallas-Bonke

, et al. Complement C5a induces renal injury in diabetic kidney disease by disrupting mitochondrial metabolic agility. Diabetes, 2020; 69(1):83–98; doi: 10.2337/db19-0043

27.

Gerl

, Bohl

, Pitz

, et al. Extensive deposits of complement C3d and C5b-9 in the choriocapillaris of eyes of patients with diabetic retinopathy. Invest Ophthalmol Vis Sci, 2002; 43(4):1104–1108; PMID: 11923252.

28.

Kheiralla

, Maklad

, Ashour

, et al. Association of complement C3 and interleukin-1 with foot infections in diabetic patients. Eur J Microbiol Immunol (Bp), 2012; 2(3):220–230; doi: 10.1556/EuJMI.2.2012.3.8

29.

Guo

, Philbrick

, An

, et al. Response gene to complement 32 (RGC-32) in endothelial cells is induced by glucose and helpful to maintain glucose homeostasis. Int J Clin Exp Med, 2014; 7(9):2541–2549.

30.

Steffen

, Tang

, Lutsey

, et al. Proteomic analysis of diabetes genetic risk scores identifies complement C2 and neuropilin-2 as predictors of type 2 diabetes: The Atherosclerosis Risk in Communities (ARIC) Study. Diabetologia, 2023; 66(1):105–115; doi: 10.1007/s00125-022-05801-7

31.

Shanaki

, Shabani

, Goudarzi

, et al. The C1q/TNF-related proteins (CTRPs) in pathogenesis of obesity-related metabolic disorders: Focus on type 2 diabetes and cardiovascular diseases. Life Sci, 2020; 256:117913; doi: 10.1016/j.lfs.2020.117913

32.

Uesugi

, Sakata

, Nangaku

, et al. Possible mechanism for medial smooth muscle cell injury in diabetic nephropathy: Glycoxidation-mediated local complement activation. Am J Kidney Dis, 2004; 44(2):224–238; doi: 10.1053/j.ajkd.2004.04.027

33.

Orchard

, Virella

, Forrest

, et al. Antibodies to oxidized ldl predict coronary artery disease in type 1 diabetes: A nested case-control study from the Pittsburgh epidemiology of diabetes complications study. Diabetes, 1999; 48(7):1454–1458; doi: 10.2337/diabetes.48.7.1454

34.

Fortpied

, Vertommen

, Van Schaftingen

. Binding of mannose-binding lectin to fructosamines: A potential link between hyperglycaemia and complement activation in diabetes. Diabetes Metab Res Rev, 2010; 26(4):254–260; doi: 10.1002/dmrr.1079

35.

Flyvbjerg

. Diabetic angiopathy, the complement system and the tumor necrosis factor superfamily. Nat Rev Endocrinol, 2010; 6(2):94–101; doi: 10.1038/nrendo.2009.266

36.

Weinstein

, Alexander

, Zack

. A review of complement activation in SLE. Curr Rheumatol Rep, 2021; 23(3):16; doi: 10.1007/s11926-021-00984-1

37.

Frazer-Abel

, Kirschfink

, Prohászka

. Expanding horizons in complement analysis and quality control. Front Immunol, 2021; 12:697313; doi: 10.3389/fimmu.2021.697313

38.

West

, Kunz

, Kemper

. Complement and human T cell metabolism: Location, location, location. Immunol Rev, 2020; 295(1):68–81; doi: 10.1111/imr.12852

39.

Ding

, Li

, Liu

. A flow cytometry-based assay for the measurement of total complement activity in the serum and clinical practice. Dis Markers, 2022; 2022:4532511; doi: 10.1155/2022/4532511