CN117092343A - Kit for detecting human immunity age, human immunity age determination method, device, system and storage medium - Google Patents
Kit for detecting human immunity age, human immunity age determination method, device, system and storage medium Download PDFInfo
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Abstract
The application discloses a kit for detecting human immunity age, a method, a device, a system and a storage medium for determining human immunity age, wherein the method for determining human immunity age comprises the following steps: s1, obtaining groups X of different age groups 3 CD8 in peripheral blood samples of human populations + Primitive T cell subset proportion X 1 And the CD8 + Expression level X of specific protein ANXA1 within primitive T cell subset 2 The method comprises the steps of carrying out a first treatment on the surface of the S2, according to X 1 、X 2 And X 3 Performing multiple linear regression analysis on natural ages, and establishing to obtain a human immune age model Y=b 1 X 1 +b 2 X 2 +b 3 X 3 +C; s3, quantifying the immune age of the sample to be detected according to the human immune age model. The human immunity age determination method provided by the application is simple, quick and accurate, is beneficial to early screening and intervention of the disease risk of people of all ages, especially middle-aged and elderly people, and is worthy of clinical popularization and application.
Description
Technical Field
The application belongs to the field of medical diagnosis and biological information, and particularly relates to a kit for detecting human immunity age, a method, a device, a system and a storage medium for determining human immunity age.
Background
As the average life span of humans starts to multiply over the past 200 years, 38% increase in the population of the elderly in the next decade worldwide will occur, and aging has become a significant health problem that cannot be ignored. Researchers generally define aging as the decline in function that occurs over time, as reflected in the cells of a variety of solid organs and tissues. Recent research into senescence has shifted from recognizing the senescent phenotype to finding potential biological pathways such as DNA damage, stem cell failure, etc. Wherein, the immune system function change is closely related to the human aging, is continuously stimulated by the internal and external environment, promotes the chronic inflammation state of the organism together with various oxidative stress factors, directly or partially causes pathological phenomena of easy infection, poor reactivity to vaccines, occurrence of nervous system degenerative change, tumor occurrence and development and the like of the old, and is an important measurement index of the human aging.
However, the immune system status of an individual does not correspond exactly to its actual biological age (natural age), experiences infections such as CMV virus, etc. that cause varying degrees of "remodeling" of the immune system, and immune aging is more likely to shorten the healthy life (health span) of the body before chronic or disabling lesions appear. Therefore, how to find immunological specific indexes to indicate the age of immunity, judge the aging of immunity and define the functional state of the immune system is critical to the health problem of the aging society, which can help us to intervene on the cellular level changes possibly occurring in the immune system at each age, thereby actively preventing the health problem related to the age.
At present, although there are few methods for evaluating the physiological age of the immune system of a human body, the immune system state is mostly judged by measuring the proportion of immune cell subsets, however, the number index alone cannot well reflect the specific function change of cells, and we need to mine more accurate biological markers and novel immune age determination methods to indicate the immune age.
T cells, which are key components of the immune system, originate in the bone marrow, differentiate and colonize the periphery after maturation within the thymus, and participate in the recycling process to exert antimicrobial infection and anti-tumor cell effects. Wherein, CD8 + T-cell (CD 8) + Primitive T cells) undergo a dual signal stimulus activation, resulting in a multifaceted change in cell cycle, metabolism and protein expression, differentiating it into effector and memory subtypes. Thus, CD8 as a key initiation part of the adaptive immune response + The state of T cells affects whether they can be activated normally and perform subsequent immune functions.
Annexin A1 (ANXA 1) is an Annexin superfamily member encoded by the ANXA1 gene at the 21.13 site of human chromosome 9 and can be expressed in Ca 2+ In the presence of the polypeptide, acidic phospholipid is combined with high affinity, and the polypeptide is expressed in various immune cells such as T cells and the like and participates in glucocorticoid-induced immunoregulation process.
At present, CD8 is not seen + Reporters of primitive T cells and ANXA1 protein to indicate human immune age.
Disclosure of Invention
The application aims to provide a kit for detecting human immunity age, and a method, a device, a system and a storage medium for determining human immunity age, so as to improve the accuracy and the high efficiency of human immunity age detection.
In order to solve the above problems, the present application provides a method for determining the immune age of a human body, comprising the following steps:
s1, obtaining groups X of different age groups 3 CD8 in peripheral blood samples of human populations + Primitive T cellsProportion of subgroups X 1 And the CD8 + Expression level X of specific protein ANXA1 within primitive T cell subset 2 ；
S2, X obtained according to step S1 1 、X 2 And X 3 And (3) performing multiple linear regression analysis on the natural age to establish a human immunity age model.
S3, quantifying the immune age of the sample to be detected according to the human immune age model;
the human immunity age model is Y=b 1 X 1 +b 2 X 2 +b 3 X 3 +C; wherein Y represents the immune age, b 1 、b 2 、b 3 Is a coefficient value and C is a constant term.
Preferably, X is the time when the subject to be examined is 0 < 18 years old 3 =1; when the age is more than 18 and less than or equal to 34, X 3 =2; when the age is 34 to 60, X 3 =3; when the age is more than 60 and less than or equal to 75, X 3 =4; x when age > 75 years old 3 ＝5。
CD8 + Primitive T cell subset proportion X 1 Finger means: CD8 + Cell numbers of the primitive T cell subpopulations are a percentage of peripheral blood.
In another aspect, the present application provides a device for determining the immune age of a human body, the device comprising:
a data acquisition module for acquiring age group X of the current subject 3 CD8 in peripheral blood samples + Primitive T cell subset proportion X 1 And the CD8 + Expression level X of specific protein ANXA1 within primitive T cell subset 2 ；
An immune age determination module for determining the X obtained by the data acquisition module 1 、X 2 And X 3 Data input the human immune age model described previously: y=b 1 X 1 +b 2 X 2 +b 3 X 3 +c to obtain the immune age of the subject.
In another aspect, the present application provides a system for determining the immune age of a human body, comprising: a memory, a processor, and a computer program stored on the memory and executable on the processor; wherein the processor, when executing the computer program, implements a human immune age determination method as described in any one of the preceding claims.
Preferably, the system further comprises: receiving newly added sample data; and updating the human immunity age model according to the stored sample data and the newly added sample data so as to improve the accuracy of the human immunity age determining system in outputting the immunity age.
Preferably, the system further comprises: the degree of aging of the immune system of the subject is determined based on the difference between the natural age and the immune age of the subject, and the degree of health of the immune system of the subject is determined.
It will be appreciated that for a fully healthy person, particularly a healthy person who is not an infant, the immune age should be less than or equal to the natural age, and if the immune age of the subject is greater than its natural age, it is indicative that the immune status of the subject is poor. Wherein, the difference between the immunization age and the time age can be expressed as: age difference = immune age-natural age. When the age difference is greater than plus 5 years old, an early warning range requiring additional attention is defined.
In another aspect, the present application provides a computer readable storage medium storing a computer program for executing the human immune age determination method according to any one of the preceding claims.
In another aspect, the present application provides a kit for detecting the immune age of a human, the kit comprising:
at least two of CD62L, CD RA and CCR7 monoclonal antibodies and ANXA1 monoclonal antibodies;
preferably, the kit further comprises: CD3, CD4 and CD8 monoclonal antibodies.
Furthermore, the monoclonal antibodies are respectively marked with fluorescein suitable for a flow cytometer, and the fluorescein marked by different monoclonal antibodies has different colors.
Preferably, the fluorescein comprises: fluorescein Isothiocyanate (FITC), allophycocyanin (APC), the complex dye PerCP-Cy5.5, 4', 6-diamidino-2-phenylindole (DAPI).
In another aspect, the present application provides a method for detecting the immune age of a human body using the kit according to any one of the preceding claims, comprising the steps of:
(1) Detecting a peripheral blood sample of a subject using the kit of any one of the preceding claims using a flow cytometer to obtain CD8 in the sample + Primitive T cell subset proportion X 1 And the CD8 + Expression level X of specific protein ANXA1 within primitive T cell subset 2 The method comprises the steps of carrying out a first treatment on the surface of the Obtaining an age group X to which the subject belongs 3 Data;
(2) X obtained in step 1 1 、X 2 And X 3 Data input the human immune age model described previously: y=b 1 X 1 +b 2 X 2 +b 3 X 3 +c to obtain the immune age of the subject.
Compared with the prior art, the application has the beneficial effects that:
(1) The application first adopts the detection of CD8 + Primitive T cell subset proportion and CD8 + Expression level of ANXA1 protein in primitive T cell subpopulations to assess human immune age, wherein CD8 + Primitive T cells are responsible for CD8 + The application discloses a starting cell with T cells playing immune effect function, ANXA1 protein can influence the immune response capacity of the T cells and the in vivo inflammation level, and the two detection indexes are closely related to the immune age through the research of the application. Thus, only total CD8 is measured relative to the prior art + The method of the application can evaluate the immune state of the organism more accurately and intuitively.
(2) The human immunity age determination method provided by the application is simple, the constructed human immunity age model has good stability and obvious correlation of detection indexes, and the accurate assessment of the immunity age of the testee can be realized.
(3) The human immunity age determining device, system or kit and the like provided by the application can rapidly and accurately determine the human immunity age only by collecting peripheral blood through veins, and the technology is mature.
Drawings
FIG. 1 is a graph showing the results of analysis of human immune cell populations and marker expression in example 1;
a represents a human immunocyte cluster map in which CD8 is highlighted + A subpopulation of primitive T cells;
b represents CD8 + mRNA expression results of primary T cell subset surface markers CD62L and CCR 7;
FIG. 2 shows that the ANXA1 protein is present in CD8 + Expression within the primitive T cell subpopulation;
FIG. 3 is CD8 of example 2 + Primitive T cell subsets and ANXA1 protein expression levels therein varied with age;
a represents CD8 + Schematic of primary T cell subpopulations ratios;
b represents CD8 + A statistical plot of the proportion of primitive T cell subsets;
c represents a horizontal flow schematic diagram of ANXA1 protein expression;
d represents a statistical plot of ANXA1 protein expression levels;
FIG. 4 is a graph showing the X in the human immune age model according to the present application 1 、X 2 、X 3 And Y pearson correlation analysis;
FIG. 5 is a graph showing the results of the fitness analysis of the human immune age model in example 2 of the present application;
FIG. 6 is a typical data presentation diagram in example 3;
fig. 7 is a graph showing the calculation results of all the samples in example 3.
Detailed Description
The technical scheme of the application will be further described with reference to the accompanying drawings and examples. However, it will be readily understood by those skilled in the art that the description of the embodiments is provided for illustration and explanation of the present application only and is not intended to limit the application as described in detail in the claims. Unless otherwise indicated, reagents, methods and equipment employed in the present application are conventional methods and test materials used, unless otherwise indicated, are available from commercial companies.
As described above, in order to mine more accurate biological markers and a novel immune age determination method to indicate immune age to improve the accuracy and efficiency of immune age detection, the present inventors have conducted intensive studies to find for the first time: (1) CD8 in peripheral blood of healthy people with normal and basal-free metabolic diseases + The proportion of primitive T cell subsets decreased with increasing natural age and were significantly related; (2) Further, annexin A1 (Annexin A1 or ANXA 1) is found in CD8 in peripheral blood of healthy people with normal, basal-free metabolic diseases + Expression levels within primitive T cell subsets rise with increasing natural age and are markedly related; annexin A1 can be used as a protein marker for indicating the immune age state of a human body.
It should be noted that, in the process of constructing the human immune age model, since the study-participating population is healthy population with normal and no basal metabolic diseases, the natural ages of these populations are defaulted to be equivalent to the immune ages in the art, in other words, the natural ages of the screened population participating in the model construction represent the immune ages.
Based on the above findings, the present inventors have further studied and validated that a method for determining the immune age of a human body is first provided, comprising the steps of:
s1, obtaining groups X of different age groups 3 CD8 in peripheral blood samples of human populations + Primitive T cell subset proportion X 1 And the CD8 + Expression level X of specific protein ANXA1 within primitive T cell subset 2 ；
S2, X obtained according to step S1 1 、X 2 And X 3 And (3) performing multiple linear regression analysis on the natural age to establish a human immunity age model.
S3, quantifying the immune age of the sample to be detected according to the human immune age model;
the human immunity age model is Y=b 1 X 1 +b 2 X 2 +b 3 X 3 +C; wherein Y represents the immune age, b 1 、b 2 、b 3 Is tied in a way thatThe numerical value, C, is a constant term.
Preferably, X is the time when the subject to be examined is 0 < 18 years old 3 =1; when the age is more than 18 and less than or equal to 34, X 3 =2; when the age is 34 to 60, X 3 =3; when the age is more than 60 and less than or equal to 75, X 3 =4; x when age > 75 years old 3 ＝5。
Further, based on the human immunity age method and the human immunity age model, the application also provides a human immunity age determining device, a human immunity age determining system and a computer readable storage medium.
In some embodiments, the human immune age determination device comprises:
a data acquisition module for acquiring age group X of the current subject 3 CD8 in peripheral blood samples + Primitive T cell subset proportion X 1 And the CD8 + Expression level X of specific protein ANXA1 within primitive T cell subset 2 ；
An immune age determination module for determining the X obtained by the data acquisition module 1 、X 2 And X 3 Data input the human immune age model described previously: y=b 1 X 1 +b 2 X 2 +b 3 X 3 +c to obtain the immune age of the subject.
In some embodiments, the human immune age determination system comprises: a memory, a processor, and a computer program stored on the memory and executable on the processor;
wherein the processor, when executing the computer program, implements the human immune age determination method described previously.
In other embodiments, the system further comprises: receiving newly added sample data; and updating the human immunity age model according to the stored sample data and the newly added sample data.
In some embodiments, the computer readable storage medium stores a computer program for performing the method of determining the immune age of a human body as described in any one of the preceding claims.
Based on the foregoing, it will be appreciated that those skilled in the art will clearly understand that the present application may be implemented by means of software and necessary general purpose hardware, and of course also by means of hardware, but in many cases the former is a preferred embodiment. Based on such understanding, the technical solution of the present application may be embodied essentially or in a part contributing to the prior art in the form of a software product, which may be stored in a computer readable storage medium, such as a floppy disk, a Read-Only Memory (ROM), a FLASH Memory (FLASH), a hard disk or an optical disk of a computer, etc., and include several instructions for causing a computer device (which may be a personal computer, a server, or a network device, etc.) to perform the method for determining the immune age of a human body according to the embodiments of the present application.
It should be noted that, in the embodiment of the device for determining the immune age of the human body, each module is only divided according to the functional logic, but not limited to the above division, so long as the corresponding function can be implemented, and the protection scope of the present application is not limited.
In another aspect, the present application provides a kit for detecting the immune age of a human, the kit comprising:
at least two of CD62L, CD RA and CCR7 monoclonal antibodies and ANXA1 monoclonal antibodies;
in some embodiments, the kit further comprises: CD3, CD4 and CD8 monoclonal antibodies.
Preferably, the monoclonal antibodies are respectively marked with fluorescein suitable for a flow cytometer, and the fluorescein marked by different monoclonal antibodies has different colors.
In some embodiments, the fluorescein comprises: fluorescein Isothiocyanate (FITC), allophycocyanin (APC), the complex dye PerCP-Cy5.5, 4', 6-diamidino-2-phenylindole (DAPI).
Further, the application also provides a method for detecting the immune age of a human body by adopting the kit, which comprises the following steps:
(1) Using flow cytometry, using the reagent of any one of the preceding claimsDetecting peripheral blood sample of the detected person by the box, and obtaining CD8 in the sample + Primitive T cell subset proportion X 1 And the CD8 + Expression level X of specific protein ANXA1 within primitive T cell subset 2 The method comprises the steps of carrying out a first treatment on the surface of the Obtaining an age group X to which the subject belongs 3 Data;
(2) X obtained in step 1 1 、X 2 And X 3 Data input the human immune age model described previously: y=b 1 X 1 +b 2 X 2 +b 3 X 3 +c to obtain the immune age of the subject.
The following will explain the research process and verification of application value of the present application in detail by examples and drawings:
example 1 screening and determination of immune age assessment index
1. Collecting clinical samples
A total of 170 volunteer blood samples were collected from a first people hospital in Shanghai city. Group entry criteria: (1) full age group; (2) healthy people without basal metabolic disease in physical examination or clinical normal examination.
2. The immune cell population of the sample is extracted, and the change data of each subgroup with age is obtained by using a bioinformatics method of single cell transcriptome sequencing.
The results are shown in FIG. 1: after the sample is processed by the SEURat program package, CD8 is clearly displayed + The presence and localization of primitive T cell subsets (a of fig. 1); and CD8 + The primitive T cell population was distinguished from other functional subsets in a defined manner by the characteristic high expression of CD62L, CCR7 surface protein markers (B of fig. 1); more importantly, CD8 + The quantitative proportion of primitive T cell subsets in peripheral blood decreases with age.
3. Based on the findings, CD8 was sequenced by single cell transcriptome + Protein expression in the original T cell subset is analyzed and mined, and differential protein indexes related to immune ages are searched.
The results are shown in FIG. 2: CD8 + Within the primitive T cell subset, mRNA expression levels of ANXA1 gene appear to be significantly progressive with ageState (a of fig. 2); in addition, data information of mRNA level of the ANXA1 gene within each age group was shown in a violin chart (B of FIG. 2) and in a bubble chart (C of FIG. 2) by split. By program statements, and the results all showed that ANXA1 was in CD8 + Expression levels within primitive T cell subsets can be a biological marker that indicates age and aging.
Example 2 method for determining human immune age and establishment of human immune age model
Based on the findings of example 1, a human immune age determination method and establishment of a human immune age model were further performed by blood samples of 170 volunteers in example 1.
(1) Peripheral blood samples of 170 volunteers are collected in an anticoagulant tube, diluted by adding equal volume of PBS, and subjected to gradient centrifugation through ficoll solution to obtain a total monocyte group in the peripheral blood. Cells were washed 2 times with staining buffer and stained by adding the desired cell surface protein antibody.
The staining antibodies include anti-human CD3, anti-human CD8, anti-human CD4, anti-human CD45RA, anti-human CD62L, anti-human CCR7, anti-human ANXA1, DAPI. Each staining antibody is coupled with a fluorescent group for flow cytometry detection, and the voltage of each color channel is adjusted.
(2) Performing flow cytometry on the stained cell sample to determine CD4 + T cell population ratio, CD8 + T cell population ratio, CD8 + Primitive T cell population proportion, CD8 expressing ANXA1 protein + The proportion of primitive T cell subsets and the protein expression intensity are input into analysis software for processing, and analysis results are shown in graphs A to D in FIG. 3.
From the above analysis, 170 samples of CD8 were obtained + Proportion X of primitive T cell subset to the number of all monocytes in peripheral blood 1 CD8 + Intensity X of expression of ANXA1 protein within primitive T cell subset 2 Meanwhile, assigning X to 170 samples according to different age groups to which the samples belong 3 : wherein 30 volunteers with ages of 0 < 18 years old are taken as X 3 =1; 45 volunteers with ages less than 18 and less than or equal to 34 take X 3 =2; 49 volunteers with ages of 34 < 60 or less are taken as X 3 =3; 21 volunteers with ages of 60 < 75 and X 3 =4; 25 volunteers with age > 75 years old, take X 3 ＝5。
(3) X obtained according to step (2) 1 、X 2 And X 3 And (3) performing multiple linear regression analysis on the natural age to establish a human immunity age model.
The human immunity age model is Y=b 1 X 1 +b 2 X 2 +b 3 X 3 +C; wherein Y represents the immune age, b 1 、b 2 、b 3 Is a coefficient value and C is a constant term. In this embodiment, b 1 ＝－0.065；b 2 ＝0.004、b 3 ＝17.727，C＝－7.218。
In this example, pearson correlation analysis was also performed on each evaluation index in the model and the immune age Y. As a result, as shown in FIG. 4, the evaluation index X set in the present application 1 (CD8 + Primitive T cell subpopulation ratio, X 2 (CD8 + Primitive T cell subset proportion, age group X 3 Has good linear correlation with the immune age Y of the sample, and has significant difference with the immune age (P<0.001)。
Meanwhile, the fitting degree of the regression model is analyzed, and as shown in fig. 5, the model constructed in the embodiment is well fitted according to the values of the R side, the standard error, the F variation, the degree of freedom of the model and the total degree of freedom.
Example 3
In this example, data of 20 volunteers ((1) full-age group, (2) healthy population without basic metabolic disease in physical examination or clinical normal examination) were additionally collected, and a human immunity age model constructed based on the application example 2 was verified, according to sample X 1 、X 2 Numerical values of (fig. 6 shows partial data analysis results) and age group X 3 The immune age was calculated and the results showed that the immune age of these 20 volunteers was consistent with their actual natural age stage (fig. 7).
To sum up, the applicationFlow cytometer to detect sample CD8 by taking a peripheral blood sample + Primitive T cell subset proportion and CD8 + The expression level of the ANXA1 protein in the original T cell subgroup, and the immune age of the subject is determined according to the human immune age model provided by the application, and the aging degree of the immune system of the subject can be determined according to the difference between the immune age and the time age after the immune age is determined, so that the health state of the immune system of the subject is determined, and the screening and the intervention of the diseased risk of people of all ages, especially middle-aged and elderly people, can be facilitated. The human immune age determination method provided by the application is simple, quick and accurate, and is worthy of clinical popularization and application.
While the present application has been described in detail through the foregoing description of the preferred embodiment, it should be understood that the foregoing description is not to be considered as limiting the application. Many modifications and substitutions of the present application will become apparent to those of ordinary skill in the art upon reading the foregoing. Accordingly, the scope of the application should be limited only by the attached claims.
Claims (10)
1. A method for determining the immune age of a human, comprising the steps of:
s1, obtaining groups X of different age groups 3 CD8 in peripheral blood samples of human populations + Primitive T cell subset proportion X 1 And the CD8 + Expression level X of specific protein ANXA1 within primitive T cell subset 2 ；
S2, X obtained according to step S1 1 、X 2 And X 3 Performing multiple linear regression analysis on the natural age to establish a human immunity age model;
s3, quantifying the immune age of the sample to be detected according to the human immune age model;
the human immunity age model is Y=b 1 X 1 +b 2 X 2 +b 3 X 3 +C; wherein Y represents the immune age, b 1 、b 2 、b 3 Is a coefficient value and C is a constant term.
2. The method for determining the immune age of a human body according to claim 1, wherein X is the time when the age of 0 < 18 years of the subject to be examined 3 =1; when the age is more than 18 and less than or equal to 34, X 3 =2; when the age is more than 34 and less than or equal to 60,
X 3 =3; when the age is more than 60 and less than or equal to 75, X 3 =4; x when age > 75 years old 3 ＝5。
3. A human immune age determination device, the device comprising:
a data acquisition module for acquiring age group X of the current subject 3 CD8 in peripheral blood samples + Primitive T cell subset proportion X 1 And the CD8 + Expression level X of specific protein ANXA1 within primitive T cell subset 2 ；
An immune age determination module for determining the X obtained by the data acquisition module 1 、X 2 And X 3 Data input the human immune age model of claim 1: y=b 1 X 1 +b 2 X 2 +b 3 X 3 +c to obtain the immune age of the subject.
4. A human immune age determination system, comprising: a memory, a processor, and a computer program stored on the memory and executable on the processor;
wherein the processor, when executing the computer program, implements the human immune age determination method according to claim 1 or 2.
5. The human immune age determination system of claim 4, wherein said system further comprises:
receiving newly added sample data;
and updating the human immunity age model according to the stored sample data and the newly added sample data.
6. A computer-readable storage medium, wherein the computer-readable storage medium stores a computer program for performing the human immune age determination method of claim 1 or 2.
7. A kit for detecting the immune age of a human, said kit comprising: at least two of CD62L, CD RA and CCR7 monoclonal antibodies and ANXA1 monoclonal antibodies.
8. The kit for detecting the immune age of a human according to claim 7, wherein the kit further comprises: CD3, CD4 and CD8 monoclonal antibodies.
9. The kit for detecting the immune age of a human body according to claim 7 or 8, wherein the monoclonal antibodies are respectively labeled with luciferin suitable for a flow cytometer, and the luciferins labeled with different monoclonal antibodies are different in color from each other.
10. A method for detecting the immune age of a human body using the kit according to any one of claims 7 to 9, comprising the steps of:
(1) Detecting a peripheral blood sample of a subject using the kit of any one of claims 7-9 using a flow cytometer to obtain CD8 in the sample + Primitive T cell subset proportion X 1 And the CD8 + Expression level X of specific protein ANXA1 within primitive T cell subset 2 The method comprises the steps of carrying out a first treatment on the surface of the Obtaining an age group X to which the subject belongs 3 Data;
(2) X obtained in step 1 1 、X 2 And X 3 Data input the human immune age model of claim 1: y=b 1 X 1 +b 2 X 2 +b 3 X 3 +c to obtain the immune age of the subject.
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