The role of the T lymphocytic cell cycle and an autogenous lymphocytic factor in clinical medicine

Bertie B. Griffths*, William J. Rea, Bradley Griffiths and Y. Pan – EHC-D Analytical Laboratory, Environmental Health Center - Dallas, 8345 Walnut Hill Ln., Suite 240, Dallas, Texas 75231, U.S.A. (*Reprint address)

Key words: cell cycle, flow cytometry, T lymphocytes, autogenous lymphocytic factor, chemical sensitivity

Abstract

In this study 315 individuals (25 controls, 290 chemically sensitive immuno-compromised patients) were investigated. Each patient had been on a standard therapy of avoidance of pollutants, nutritional supplementation, and injections of antigens for foods, and biological inhalants, but did not attain their immunological competence. Peripheral lymphocytes were collected and DNA histograms were constructed. The flow cytometer was used to evaluate the cell cycle, hematological, and other immunological profiles. From the other portion of the blood specimen, lymphocytes were propagated in vitro, harvested, and a lysate, termed the autogenous lymphocytic factor (ALF), was prepared. When treated with ALF, 88% of these individuals showed a significant (p<0.001) clinical improvement which correlated with laboratory findings, involving regulation of abnormal cells cycles, increase in total lymphocytes and subsets T4'T8' (p<0.05) and cell mediated immunity (CMI) response (p <0.001). The ALF presumably acts as a biological response modifier. The cell cycle and ALF provide clinical tools for diagnosis and regulation of immunological incompetence.

 

 

Introduction

The cell cycle is the ordered and orderly events of biochemical and morphological sequences, leading from the formation of a daughter cell as a result of mitosis, to the completion of the processes required for its own division into two daughter cells (Lee and Dang, 1995). It is fundamental that following mitosis, two daughter cells are produced. These cells may initiate a new cycle (G1 phase), some cells may become non-proliferative (G0 phase) or progress to a restriction point where they are committed to the synthesis of cellular components, (the S phase) and finally complete the cycle (the G2M phase). The cell cycle then comprises the sum of the growth phases of a specific cell cycle. This cycle is repeated by continuously dividing cells. Even within the same organism the specific cell cycle will vary with different classes of cells. The total time that is necessary to complete the S and G2 phases is generally constant in different cell types. It seems reasonable then, to assume that most of the time variation takes place in the G1 phase.

To establish homeostasis, it is imperative that the cell cycle be regulated. To this end, there are biological parameters which may be employed to ascertain the normality of a particular cell cycle. The DNA content in the nucleus of a cell (2 N or diploid amount) is constant in all normal organisms (Givan, 1992). There are only two exceptions where the DNA content is not constant; the amount of DNA will vary in cells which undergo meiosis in preparation for sexual reproduction, thus containing the 1 N or haploid amount of DNA. The other exception applies to those cells which undergo DNA synthesis in preparation for mitosis. These will contain between 2 N and 4 N amounts of DNA.

By employing the concept of DNA constancy in a particular organism, and the application of flow cytometric techniques, DNA flow histograms can be constructed depicting a normal cell cycle and the identification of abnormal cycles.

The regulation of the cell cycle in eukaryotes seems to take place at two main transition points, prior to DNA replication at a point in the G1 phase, termed the restriction point, and prior to cytokinesis at the G2M phase boundary (Nurse and Bissett, 1981). The progression of the cell cycle from one phase to the other is mediated by specialized proteins, cyclins, and the activation of enzymes called cyclin-dependent kinases (CDKs) and by a number of positive and negative feedback loops. To date seven CDKs have been identified and designated CDK 2 to 8 (Meyerson et al., 1992; Lorinez and Reed, 1984; Nurse, 1994; Sherr, 1994; Helchman and Roberts, 1994; King et al., 1994; Hunter and Pines, 1994). Each CDKs acts at different stages of the cell cycle and is differentially regulated by different cyclins (Meyerson and Harlow, 1994). The critical roles played by cyclins in the regulation of eukaryotic cell cycle is amply documented.

These CDKs play very significant roles in the G1S and G2M transitions during the mammalian cell cycle. They regulate by phosphorylation, a number of key substrates which subsequently activate a transition from G1 to S and from G2 to M (Kamp et al., 1994). The catalytic subunits alone of these CDKs are not active and require the influence of positive regulatory subunits to ensure biochemically active protein kinase holoenzymes (Desai et al., 1992). The positive regulatory subunits employed to this end are cyclins. The activity of CDKs is, therefore, regulated by both cyclins and specific phosphorylation and de-phosphorylation (Gold and Nurse, 1989; Draetca and Beach, 1988; Solomon et al., 1990; Gutier et al., 1991; Murray and Krischner, 1989).

Cyclins were identified originally as proteins in the murine invertebrate cells. The concentration of these cyclins accumulate and are destroyed periodically at defined points during the cell cycle (Evans et al., 1983). At present, eight cyclins have been identified; these are designated A, B, C, D, E, F, G1, and H based on their amino acid sequences (Pines and Hunter, 1989; Xiong et al., 1991), and in some instances, on genetic complementation experiments in yeast (Nurse, 1990; Murray and Krischner, 1989; Lewin, 1990; Sherr, 1993).

The influences of the cyclins are expressed differently; e.g. cyclin A exhibits its influence through the S-M phase (Wang et al., 1990; Fang and Newport, 1991; Walker and Maller, 1991; Lehner and O’Farrell, 1990; Pagano et al., 1992; Zindy et al., 1992) of most cells, while cyclin B (which is conserved from yeasts to humans) propels cells into mitosis (Nurse and Bissett, 1981; Evans et al., 1983; Glotzer et al., 1991). Both the A and B cyclins are degraded at the M phase by ubiquitin (Ub)-dependent proteolysis (Glotzer et al., 1991). G1 cyclins (CIN 1, CIN 2, CIN 3) are assumed to associate with a p34 CDC2 homologue, p34 CDC 28, driving yeast cells into S phase (Reed, 1992; Cross, 1988; Richardson et al., 1989; Hadwiger et al., 1989; Wittenberg et al., 1990; Nasmyth, 1990).

In the Environmental Health Center - Dallas (EHC-D), clinical cases are investigated varying from surgical to environmental illnesses. However, cases involving chemical sensitivity entail the principal aspect of our practice. Over a period of 22 year our laboratory observations show that characteristically, a percentage of the chemically sensitive individuals portray a secondary deficit which is related to immune dysfunction, principally, depression of the suppressor T-cell (Rea et al., 1986, 1987).

Initial profiles of lymphocyte cell cycle progression, T and B lymphocytes, cell mediated immunity and haematograms of patients were compared with those taken subsequent to treatment with autogenous lymphocytic factor (ALF). Acting presumably as a biological response modifier, ALF was observed to modulate deregulated immune profiles.

The focus of this investigation was firstly to observe the patterns of DNA histograms of the T lymphocyte cell cycles, especially in chemically sensitive individuals. The histograms provide a "snap-shot" of the individual’s present cell cycle. By comparing an individual’s cell cycle with the classical normal cycle, very crucial information will be obtained to enable a scientific regulation of a patient’s cell cycle. Secondly the work was to prepare a lysate from the in vitro propagated peripheral T lymphocytes of an individual with an with an irregular T lymphocytic cell cycle, and observe the regulatory effect of this lysate, when injected subcutaneously into such an individual. Thirdly, the intention was to establish a basis for the regulation of an individual’s T lymphocytes which were observed to be irregular due to varied incitants, thus restoring normal T lymphocyte functions, and the enabling of a compromised individual to cope with multiple insults to his/her immune system. Fourthly, we examined the responsiveness of T lymphocytes to autogenous lymphocytic factor (ALF) as measured by the cell mediated immunity (CMI) test, and to establish on an individual basis an index of T lymphocyte ability to respond.

From our search of databases, and to the best of our knowledge there is no documentation pertaining to the regulation of the T lymphocytic cell cycle by lysates of an individual’s T lymphocytes, and its application to clinical medicine.

Materials and methods

Isolation of lymphocytes

Venous blood was collected from 315 individuals in heparinized tubes. Of these 25 were healthy employees of EHC-D; these represented normal controls. The other individuals were chemically sensitive, chronically ill patients of the EHC-D and referrals from outside physicians. Erythrocytes were separated by a modified gradient technique using heparinized CPT vacutainers (Becton Dickinson Co.). This preparation was centrifuged from 30 min at 500 x g. The lymphocyte layer (interphase between isolymph and plasma) was removed to sterile polystyrene tubes. Lymphocytic layers were washed three times with normal saline (0.9% sodium chloride) by centrifugation in a refrigerated centrifuge at 500 x g for 20 min. Cells were re-suspended in normal saline and counted.

Lymphocytes phenotyping

Monoclonal antibodies against pan T cells, T11 (CD2), T helper cells T4 (CD4, T suppressor/cytotoxic cells T8 (CD8, activated T cells TA1 (CD26) and pan B cell B4 (CD19 were bought from Coulter Immunology (Hialeah, Florida). Peripheral lymphocytes were obtained by venipuncture, and stained by the use of Coulter Q prep Epics immunology station following manufacturer’s instructions.

Preparation of cell cycles

Heparinized whole blood or lymphocytes were used and 100 µ1 aliquots of heparinized whole blood or 100 µ1 of washed isolated lymphocytes (suspended in normal saline) were placed in 13 x 75 mm tubes. To each tube was added 200 µ1 of lysing buffer (Coulter) and gently mixed for ~15s, then 2.0 ml of DNA stain with RNAse was added and mixed for 20 s. The mixture was stained with propidium iodide at a concentration of 50 mg per ml, and allowed a staining period of 15 min and then analyzed by flow cytometry.

Flow micro-fluorometric analysis

Cells tagged fluorometrically for DNA content were analyzed on the Coulter Epic C flow cytometer, where they were allowed to pass through a laser beam tuned to 488 nm. Fluorescence was measured electronically and recorded as a histogram. DNA distribution in the cell cycle was calculated on accumulated data by parametric analysis to produce a particular DNA histogram.

Preparation of autogenous lymphocytic factor (ALF)

Propagation of lymphocytes

To cell culture flasks, containing 10 ml RPMI 1640 medium and/or supplemented with 1 ml bovine calf serum was added 1 ml of washed T lymphocytes. The culture was incubated at 37°C and monitored daily for approximately 2 to 3 weeks.

Harvesting of cell

When the cell population approximated 5 to 9 x 106 per ml, culture suspensions were transferred to calibrated 50 ml conical tubes and centrifuged for 30 min at 500 x g. The supernate was discarded, cell pellet re-suspended in 10 ml normal saline and washed three times by centrifugation for 30 min each time.

Preparation of ALF

Each cell pellet was re-suspended in ~2.0 ml normal saline and sonicated at 20 W with a duty cycle of 50% for 60 s. The sonicated mixture was sterilized by filtration and stored at -20°C to be diluted for therapeutic use as indicated.

Establishment of a functional T lymphocyte cell cycle

The profile of an individual’s T Lymphocyte may appear normal, yet the lymphocytes may not be functional. A process was established to evaluate the immunological responsiveness of the lymphocytes of the cell cycle.

Stimulation of peripheral lymphocytes

NunclonTM-plastic plates (24 wells) containing 2 ml of RPMI 1640 medium per well were inoculated with ~2 to 4 x 106 lymphocytes pr ml, 1.25 ng interleukin 1 alpha (Il1 alpha) per ml, or with 0.1 ml ALF per ml; negative controls had neither Il1 nor ALF. The plates were incubated at 37° for 96 h. Cells were harvested, washed and analyzed by flow cytometrically as mentioned above. Comparison of the S and G2 phases of both the un-stimulated and the stimulated T lymphocytes was undertaken and an index computed from the equation:

stimulation index = (stimulated S + G2)/(un-stimulated S + G2)

Clinical Investigation

A total of 315 individuals was investigated and of these, 25 were controls. T lymphocyte cell cycle profiles, hematological, T and B profiles, and CMI tests were carrier out on all candidates. Individuals with immune deregulation were treated with autogenous lymphocytic factor (ALF). These individuals were chemically sensitive and chronically ill. The illnesses include dermatitis, vasculitis, asthma, organic brain syndrome, and Gulf war syndrome. In all cases, these illnesses were characterized with immune system suppression, dysfunction or deregulation.

Major symptoms presented by these patients include one or more of the following symptoms: lacrimation, pruritus, swelling and puffiness (ocular); fullness, noise, and dizziness (otic); congestion, sneezing, rhinorrhoea and blowing (nasal); lump, clearing and postnasal drip (throat); hypersensitivity reactions (immune); arthritis and arthralgia, fatigue and muscle pain ( musculoskeletal); pressure and cough (chest); weight loss and fatigue (constitutional); miliary, ethmoidal and frontal (headache); insomnia, shortness of breath and depression (neurological).

All 290 chemically sensitive individuals presented a history of being affected by environmental incitants found in categories such as food, biological inhalants, and chemicals. They presented histories of varied backgrounds, but common among them was that all showed irregular cell cycles and abnormal T lymphocyte profiles in both numbers and functions in T and B lymphocytes and subsets. DNA histograms showed over or under accumulation of various subtypes of lymphocytes in one or more phases of the cell cycle of each individual.

Cell-mediated immunity

Delayed cutaneous hypersensitivity, or cell-mediated immunity (CMI) responses were evaluated in 190 patients; the results were recorded before and after treatment with ALF. The multi-test CMI test kit (Mèrieux Institute, Miami, Florida) was used. Each kit contained seven antigens: tetanus, diphtheria, candida, proteus, streptococcus, trichophyton, and tuberculin. The tests were evaluated and read at 48 h. Evaluation involves scoring the size, and number of increase of the wheals. The diameter of each induration was measured in millimeters and averaged. A reaction was considered positive if the average diameter was 2 mm or more.

Comparative evaluation of T and B lymphocytes

T and B lymphocytes and subsets were evaluated flow cytometrically before and after treatment with ALF.

Table 1 Representative profile of symptoms and signs after autogenous lymphocytic factor treatment

 

Symptoms

Improvements

No change:

Deterioration:

Total

Patients

 

 

Number

%

Number

%

Number

%

 

 

Hypersensitive reaction

63

63

33

33

1

1

100

Recurrent infection

38

57

29

43

0

0

67

Fatigue

60

68

27

31

1

1

88

Lack of concentration

43

54

36

46

0

0

79

Arthritis

19

44

23

54

1

2

43

Gastrointestinal upset

29

40

43

60

0

0

72

Headache

28

44

33

53

2

3

64

Depression

42

58

30

42

0

0

72

 

Establishment of therapeutic dosage of ALF

In vitro and in vivo procedures were used to establish dosage. Blastogenesis was the in vitro test employed for establishing dosage in general. In vitro grown peripheral lymphocytes were challenged comparatively with the known mitogens phytohaemagglutinin (PHA), concanavalin A (Con A), and pokeweed mitogen (PWM) of varied concentrations. A dose responsive curve was established to evaluate the efficacy of ALF with that of the mitogens; 0.1 ml of 1:10 dilution of ALF was found to be optimal when administered twice weekly. Intradermal test was carrier out on all recipients depending on their response and sensitivity. If unusual sensitivities were observed, the ALF was further diluted. This procedure was successively repeated until no adverse reaction was observed.

Results

Significant changes were observed within 1 to 6 weeks in patients treated with ALF. Changes were observed in immune regulation and overall clinical manifestations. With regard to clinical manifestations, there were noteworthy improvements (Table 1), although minimal symptoms continued after approximately 3 weeks of continued therapy.

In a normal cell cycle, the highest percentage of lymphocytes should be in the G0 to G1 phase (Figure 1). This percentage will change dramatically when these lymphocytes are stimulated by various incitants (Figures 3a and 3b). Consequently, different percentages will appear in the S and G2M phases, producing a deregulated profile (Figures 1 to 4). Treatment with ALF regulates the T lymphocyte cell cycle profile.

Immunologically, there were significant regulations of T lymphocyte cell cycles, especially from one phase of the cycle to another. Patients become less sensitive and more tolerant to specific incitants. As treatment continued, in general in about 6 weeks, a more drastic shift toward that of a normal profile was observed. Figure 1 to 4 summarize the regulatory changes of some of the cell cycles studied.

Changes were observed in the profiles of the T and B cells where T and B lymphocytes and their subsets were evaluated before and after treatment. There was a significant (p<0.01) change in the total lymphocyte count and subsets, T4, T8 in 92 patients investigated. It should be noted that ALF seems to act as an immune modifier since the total lymphocytes, T4, and T8 were significantly elevated or reduced in order to maintain normalization (Tables 2 and 3).

Figure 1 (Not shown)

 

Figure 1 Diagrammatic representation of a normal mammalian cell cycle, showing the relative number of lymphocytes in each phase of the cycle, as displayed by the intensity of fluorescence of the fluorochrome-bound DNA in each lymphocyte. Normally, the percentage of lymphocytes in each phase of the cell cycle is 90-95, 5-10 and 0-5 I ht G0, G1 phase, S phase and G2M phase, respectively. These quiescent lymphocytes display, normally, a low level of replication, and progression through the cell cycle. In response to stimuli, these cells undergo rapid division and cycle rapidly. The shading show that there is overlapping of phases and not specific lines of demarcation: stippled, G 0, G1 phase (90-95% non-cycling lymphocytes); hatched, S phase (5-10% cycling lymphocytes); unshaded (open), G2M phase (0-5% cycling lymphocytes); and solid areas indicate overlapping of phases.

Table 2 Representative changes in T and B cell profiles after 92 chemically sensitive patients were treated with autogenous lymphocytic factor

 

 

Treatment

Total

lymphocytes

 

T11

 

T4

 

T8

 

T4,/ T8

 

B4

Before

2,112±632

1,624±457

930±45

439±58

2.3±0.8

188±102

After

2,232±678*

1,634±544

1,027±297**

478±189**

1.3±7.4

171±120

*p<0.05; **p<0.01; n=92

 

Figure 2 (Not shown)

Figure 2 A representative DNA histogram of peripheral T lymphocytes from normal volunteers. Note that the highest percentage of lymphocytes is in the G0G1 phase, as expected, but the percentage varies in different individuals in keeping with the prevalence of environmental pollutants. Thus, the ideal normal profile is seldom achieved. The abscissa shows the relative fluorescence of the fluorochrome-bound DNA in each cell.

Table 3 Profile of lymphocyte subsets modulation after treatment with autogenous lymphocytic factor

 

After

treatment

Total

lymphocytes:

 

T11

 

T4

 

T8

 

 

Number

%

Number

%

Number

%

Number

%

Increase

52

57

41

46

53

53

55

60

Decrease

40

43

51

54

39

42

37

40

Probability

<0.05

>0.05

<0.05

<0.01

Table 4a Typical cell-mediated response in (number and size) chemically sensitive individuals treated with autogenous lymphocytic factor

 

Patients

Before ALF

After ALF

P

190

5.46 ± 5.81

9.28± 7.25

<0.001

Results are given as mean ± SD.

 

 

 

 

Patients showed significant improvement (p < 0.001) in their CMI scores (Table 4). The regulatory effect of the immune system can be objectively assessed periodically after the initial treatment with ALF. The resourceful parameters are profiles of T lymphocyte cell cycle, T and B lymphocytes, and their subsets, cell-mediated immunity, signs and symptoms.

Side effects of ALF

The side effects were minimal and occurred only in six adults (five females and one male) where the average age was 52 years. These patients were intolerant of ALF, their symptoms included pain and irritation in the throat, burning in the eyes, pain and irritation in the chest, heat palpitations, influenza-like symptoms, headache, fatigue, and chills.

Table 4b Cell-mediated immunity positive score (number and size) after treatment with autogenous lymphocytic factor

 

Score

Patients

%

P

Increase

No change

Decrease

58

9

11

74

12

14

<0.001

Figure 3 (a) and (b) (not shown)

Figure 3a A representative irregular T lymphocyte cell cycle profile showing the effect(s) of stimulating the environmentally ill patients influence, primarily, the lymphocytes in the S phase.

Figure 3b A representative irregular T lymphocyte cell cycle profile showing the effect(s) of the stimulating incitants on the lymphocytes in the G2M phase. It seems that each incitant, or a mixture of incitants affects lymphocytes in a particular phase(s) of the cell cycle, resulting in a variety of irregular cell cycle profiles and presumably dictates varied patterns of clinical manifestations. The abscissae in Figure 3a and 3b show the relative fluorescence of the fluorochrome-bound DNA in each cell.

Discussion

The flow cytometric profile of the T lymphocyte cell cycle as demonstrated by DNA histogram presents a reflection of the status of T lymphocytes in an individual. Of great importance is that its application is not limited to a certain category of individuals, but to normal subjects as well as individuals who are compromised by varied incitants. The essence of its clinico-biological importance, as detected in the present investigation, is that it is a reflection of T lymphocyte cell function, and may facilitate an in-depth approach to the treatment of some immuno-deficient illnesses.

The progression of T lymphocytes from one phase of the cell cycle to another is time-dependent, ~8 to 12 h in the G0-G1 phase, 6 to 8 h in the S phase, and 0.5 to 1 h in the G2M phase. The 290 individuals who were investigated in this study were affected principally by environmental incitants in categories such as foods, biological inhalants, and chemicals. They presented histories of varied backgrounds and different cell cycle profiles. The DNA histogram cycles showed over or under accumulation of lymphocytes in one or more phases of the cell cycle of each individual (Figures 3a and 3b). As shown in Figure 2, even the normal controls do not present an ideal DNA histogram profile of T lymphocytes. This is due to the fact that, in general, the ideal environment is seldom ever achieved, thus there is always some degree of immunological compromise to most individuals. It is reasonable then to assume that the incitant(s) which lead to immuno-incompetence of these individuals are capable of inactivating the particular enzyme(s), cyclin-dependent kinases, or depleting specific cyclins whose combination with their specific kinases are instrumental in catalyzing the progression of T lymphocytes from one phase to another of the cell cycle. The T lymphocytes of the individual affected are locked in a particular phase, either resting, synthesizing, or multiplying too much in the G0-G1,S, G2M phase respectively. Thus the subject manifests symptoms peculiar to the phase(s) affected. This hypothesis offers an opportunity to associate clinical manifestations with T lymphocyte cell cycle irregularity.

The treatment of lasting importance would be reasonably thought of as a biological response modifier, which would stimulate CDKs, regulate the cell cycle and the enzymes of purine and pyrimidine nucleotide synthesis. It is now generally accepted that these enzymes are elevated during the S phase (Cory, 1993). It seems logical that ALF stimulates these regulatory functions. The autogenous lymphocytic factor is a dialysable mixture of the many effector substances which may be released from in vitro grown stimulated lymphocytes with the ability to invoke immunological influences in vivo or in vitro, (Griffiths and Rea, 1995). The expected biological activity(s) of ALF would be to act as a biological response modifier, with mechanisms of action as suppressor and regulator of the immune system, especially in the regulation of the T lymphocyte cell cycle.

Kamp et al. (1994) showed that progression through the cell cycle requires the joint influence of positive regulator subunits, cyclins, and cyclin dependent kinases. These subunits regulate by phos-phorylation a number of key substrates which subsequently activate a transition from G1 to S, and G2 to M phases of the cell cycle. Phosphate groups are transferred from ATP to a special amino acid in the target protein by protein kinases, while phosphatases remove the phosphate groups from the target proteins. The addition and removal of phosphate groups significantly affect the biochemical behavior of the target proteins. Many protein kinases and phosphatases have a specific affinity for their target proteins, and act as determinants for controlling the activity(s) of their target proteins. Indeed, ALF may possess these protein kinases which, acting as molecular switches, regulate an irregular T lymphocyte cell cycle to that of an ordered and orderly progression.

As demonstrated (Figures 4c), ALF regulates deregulated cell cycles, improves immunological profiles by increasing or decreasing the number of circulating lymphocytes and their sub-populations (Table 2 and 3); and restores immune responses as demonstrated by enhanced cutaneous hypersensitivity (CMI) (Table 4). ALF also invoked immediate intradermal test response within 45 min of administration subsequent to the administration of antigen challenges which were previously negative or delayed for at least 15 days. Responses were observed by the number and size of wheals to the antigens which were inoculated. It is noteworthy that some chemically sensitive patients responded favorably, or completely recovered after environmental exposure to air, food, water, nutritional support and exercise. Some patients responded but did not fully recover. The patients who recovered reacted profoundly to exposure of very minute environmental insults of ambient chemicals, biological inhalants, and foods, or inhalants. However, when these patients were treated with Alf, their hypersensitivity was markedly reduced or disappeared; there was also significant improvement in recurrent infection, fatigue, headaches, depression, concentration, and gastrointestinal upsets. Eight patients immediately improved on initial injection with ALF.

T-suppressor, T-helper cells, and total lymphocytes were increased as reflected in an increased in a low population, and a decrease in a too high population. This suggests that ALF is a modulator rather than a simulator. The mechanism(s) of action of ALF have not yet been ascertained. Indeed, the purification and determination of ALF from sorted T lymphocytes with or without helper phenotypes will be necessary to facilitate understanding the mechanism of action. However, it has been documented that the human cells contain a regulatory protein, CKS protein, which is a genetic suppressor of temperature sensitive CDK mutations. There are two isoforms of this CKS protein, namely CKSH1 and CKSH2. It is believed that CKSH2 protein binds to the catalytic subunit of the CDKs, and is essential for their biological function (Parge et al., 1993). To this end, further investigations are being carried out.

The concept of a functional cell cycle is timely. Indeed, the profile of a cell cycle offers an insight into the immune status of a patient. However, the cell cycle profile does not indicate whether or not the lymphocytes are immunologically responsive or capable of responding to therapy. The influence of varied environmental incitants and/or other systemic illnesses may render these lymphocytes incapable of normal progression through the cell cycle, thus resulting in a non-functional cell cycle. In vitro lymphocyte activation represents a standard procedure for evaluating cell mediated responses to a variety of stimuli including antibodies, polyclonal mitogens, specific antigens and cytokines. Interleukin-1 alpha (Il1 alpha) and ALF were used as activators in the present investigation. Interleukin-1 alpha was used due to its extremely broad spectrum of bio-activity. ALF was used to observe its capability as a biological response modifier. The stipulatory process presented in this investigation offers a method of computing lymphocyte stipulatory response, and consequently an opportunity to establish an individual regime of treatment of illnesses associated with immunodeficiency. This investigation offers: (1) a vivid picture of the status of the immune profile of a patient as demonstrated by the DNA histogram of the T lymphocyte cell cycle; (2) an opportunity to regulate an irregular T lymphocyte cell cycle by treatment with autogenous lymphocytic factor; and (3) a clinical tool for the regulation of abnormal cell cycles and/or hematological and immunological profiles in humans with immunological deregulation. It should be noted that there is a pending United States patent, number 08/380,063 for the application of the cell cycle to clinical medicine, and also the regulation of the cycle by autogenous lymphocytic factor.

Acknowledgments:

The authors are grateful to Dr. Alfred Johnson and Dr. Gerald Ross of the Environmental Health Center - Dallas, for their contribution to the clinical data, and to Dr. Vernon E. Scholes for reviewing this manuscript.

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