**4. The role of RBC antigens transport in health and disease**

22 Blood Cell – An Overview of Studies in Hematology

subjected to lyses by freezing.

antibodies against those antigens.

**Figure 9.** Illustrates the dynamics of RBC's antigens

**Results** 

Figure 9.

contained sheep RBC hemolysate.

Infect

Infect

direct bacteria slide agglutination test.

3. Rabbits sera were separated and examined for antibodies against E. coli O157 using

5. Red blood cells were prepared from anti-coagulated sheep blood collected at 0 time (i.e., before inoculation), 1st week, 2nd week, and 3rd week. The collected blood was centrifuged at 4 C for 25 minutes at 1170 g. Plasma and Buffy coat from each sample were removed. RBC were washed twice in normal saline solution by centrifugation at 4 C for 5 minutes at 2000 g, and then re-suspended in Tris/Saline buffer pH 7.5 and

6. Nobel agar 1% in Tris/Saline was used as a supportive media for antigen-antibody precipitation, where the central well contained rabbit serum and peripheral wells

The rabbit serum showed high titer (1/160) of antibodies against E. coli. Antigens of E. coli could be precipitated from sheep RBC of the 1st and 2nd week after infection, only,

**Figure 8.** Preparing Antibodies against bacteria and preparing RBC carrying antigens of this bacteria. The purpose is to precipitate Bacteria antigens from RBC of infected animals using the prepared

> **Central well:** Rabbit antiserum **Well 1:** Sheep's RBC before infection

**Well 3:** Sheep's RBC 2nd week after infection **Well 4:** Sheep's RBC 3rd week after infection

**Well 2:** Sheep's RBC 1st

Detect antibodies against E. coli

RBC

Antiserum

week after infection

4. A sheep was infected by oral administration of bacterial suspension.

The RBC transport function maintains tolerance to self antigens. This function is exploited positively to protect a fetus from the immune system attack using the same mechanism of protecting the self. In effect, a fetus, which is an allograft, is considered part of self.

In humans and animals, not all microorganisms are capable of causing disease. Some of those microorganisms are equipped with the machinery that can overcome biological barriers and can cause disease in animals but not in humans and vice versa [15]. The role of RBC antigens transport in inducing tolerance to self-antigens is a feature that can be considered as a security-hole, as invaders can exploit this process to escape from the response of the immune system by disguising themselves as self. Tumors and parasites are negative examples.

Notice that this mechanism of tolerance induction does not contradict with all what we know about tolerance. Further, it explains the documented properties of tolerance. For instance, some of the properties that can be explained are:


Notice that the discovered function of RBC fills a gap in the understanding of tolerance. Part of this gap can be expressed in the following questions:


Answer of Question 1 and 2: RBC can easily absorb soluble antigens through pinocytosis while a particulate antigen needs receptor sites on RBC in order to be absorbed, which is the RBC membrane antigens function. Notice that the probability that the immune system will react to some processed antigens still exists. That is why the dose of antigens plays an important role. As far as there are enough stores of antigens in RBC, they are effectively tolerated.

Answer of Question 3: If antigens are introduced to a fetus while the immune system is still incapable of respond, there is a good chance for those antigens to be processed by the Antigen-Presenting-Cells (APC) and then absorbed by RBC. When mature lymphocytes production starts, later in life, antigen stores of RBC are used to induce tolerance. This may explain why tolerance is easier to induce in prenatal life.

Further, a pathogenesis mechanism of some autoimmune disease can be postulated. If RBC antigen-transport function is impaired for a particular self-antigen, for some reason, the

tolerance to that antigen will eventually vanish. Consequently, an autoimmune response will be provoked to that antigen and autoimmune disease is established.

Rediscovering Red Blood Cells: Revealing Their Dynamic Antigens Store and Its Role in Health and Disease 25

The proposed mathematical model and a data mining algorithm will not only help in identifying proteins (antigens) that can be used in diagnosis and treatment of difficult disorders, but also will help in etiological diagnosis of idiopathic disorders and their treatment. This approach is based on building large databases of RBC antigens' store for patients and normal individuals. Consequently, a patient sample is collected on anticoagulant. RBC and plasma are separated. The plasma IgG is separated and then used as ligand in immunoaffinity chromatography to separate hemolysate antigens. The collected antigens are identified by mass spectrometry. The database record consists of the diagnosis

It consists of four main parts; definitions of symbols, model of diseases caused by microorganisms, tumors, or foreign proteins; model of autoimmune diseases which result as a consequence of missed tissue proteins from RBC antigens' store; and model of diseases of

Op: (pi , dj), ordered pair of patient presented by protein sequence (i) and health state (j).

Pdj = ∩ {Pp}dj

Pnormal = ∪ {Pp}normal

P'normal such that p in Pnormal if the number of occurrence of p Pnormal is less than 5% of the

P'dj = Pdj – P'normal

Where P'dj is the set which contains proteins that can be used as biomarker or vaccines,

a. Model of Diseases caused by microorganisms, tumors, or foreign proteins

Where Pdj is the set which contains all common proteins associated with dj.

Where Pnormal is the set which contains proteins associated with normal.

**5.2. Bioinformatics approach for products development** 

and the set of identified antigens.

*5.2.1. Mathematical model* 

unknown cause (Idiopathic).

D = {d1, …, dm}, Set of all diseases

Let the assumption of this work be as the following: pi: protein amino acid sequence, where i = 1 .. n

dj: health state, i.e., normal or disease name, where j = 1 .. m P = {p1, …, pn}, Set of all proteins of RBC antigens' store

Pp: patient proteins where Pp P where p is the patient ID

total number of p in Pnormal then remove p from Pnormal.

**Definitions** 

Figure 10.
