in particular , referring to the single autoimmune disease above said and to results of in vitro and in vivo experiments reported herein after , preferred daily administration of apotf are : multiple sclerosis : therapeutic regimen at ≧ 2 . 5 mg / kg body weight ; rheumatoid arthritis : prophylactic and therapeutic regimen at ≧ 1 mg / kg body weight ; intestinal inflammatory diseases : prophylactic and therapeutic regimen at ≧ 5 mg / kg body weight ; diabetes mellitus type 1 : therapeutic regimen at ≧ 1 mg / kg body weight ; autoimmune hepatitis : prophylactic and therapeutic regimen at ≧ 2 . 5 mg / kg body weight . the present invention is easier to understand in the light of the following examples of embodiments . we assessed the capacity of apotf to modify certain functional parameters in vitro of lymphomonocytes of various type and origin , such as their proliferative response to mitogens and the production of pro - inflammatory cytokines and nitrites . for the assessment of lymphomonocytic proliferation , a method was developed to separate the lymphomonocytes obtained from cba and c57bl mouse spleens . the spleens were homogenized by passing them through a fine - mesh screen and subsequently submitted to centrifugation at 350 g for 5 minutes . the resulting cell pellet was resuspended in rpmi with 5 % of fcs , placed in a test tube , covered with ficoll solution and centrifuged at 550 g for 18 minutes . the resulting ring of lymphomonocytes was collected from the ficoll medium , washed twice to remove the residual ficoll , resuspended in growth medium ( a mixture of rpmi 1664 , antibiotics and antimycotics , and 5 % of fcs ), and transferred to 96 - well plates at a concentration of 500 , 000 cells / well . each cell line , with or without mitogenic stimulation with cona 2 . 5 μg / ml , was cultured without the drug or with one of three different concentrations of apotf ( 6 . 25 μg , 12 . 5 μg , 25 μg per ml ). the incubation period was protracted for 3 days , after which 1 μci of tritiated thymidine was added to the cells . the residual radioactivity after 18 hours , established from a beta - counter reading , was considered as an indicator of proliferative response . the results obtained from different sets of experiments , expressed as beats per minute ( bpm ), are shown in fig1 and 2 . for the purposes of evaluating tnf - α and il - 2 secretion , splenic mononuclear cells from cba , balb / c and c57bl mice obtained as described previously were placed in standard growth medium at concentrations of 5 × 106 and 1 × 107 cells / well for a period of 48 hours , with or without mitogenic stimulation ( cona 2 . 5 μg / ml ) and with or without transferrin ( 12 . 5 μg / ml ). the tnf - α and il - 2 were measured using the elisa test . the sensitivity limit for these tests was 15 pg / ml . the results obtained are shown in fig3 a and 3b . to assess the effects of the drug in question on the production of nitrites , splenic mononuclear cells from balb / c mice , obtained as described previously , were placed in standard medium at concentrations of 5 × 106 and 1 × 107 cells / well , with or without mitogenic stimulation ( cona 2 . 5 μg / ml ), and without the drug or with one of three different concentrations of apotf ( 6 . 25 , 12 . 5 , 25 μg / ml ) for an incubation period of 48 hours . the concentrations of no in the splenic mononuclear cell cultures were identified by colorimetric assay exploiting the griess reaction , which is able to determine the quotient of nitrites in the cell surnatant , with or without mitogenic stimulation ( cona 2 . 5 mg / ml ). for this purpose , in particular , 50 ml of griess reagent were added to 50 ml of surnatant and , after 10 minutes , the consequent colorimetric reaction was measured with an elisa reader at 570 nm . the immunomodulatory capacity of the molecule was also tested on peritoneal lymphomonocytes from cba , balb / c and c57bl mice . briefly , peritoneal cells were isolated from the abdominal cavity of cba , balb / c , c57bl mice by means of an intraperitoneal injection of 1 ml of pbs , followed by the collection by aspiration of the whole quantity of pbs , including the peritoneal cells , which was placed in a test tube and centrifuged at 350 g for 5 min . then the cells were counted and placed in complete growth medium ( rpmi 1640 [ gibco ], with 200 mm of 1 % l - glutamine , 10000 u / m of 1 % penicillin - streptomycin and 5 % fcs ) in 24 - well plates . two different sets of cell cultures were prepared to determine the levels of tnf - α : cba mouse peritoneal cells were placed in a culture at a mean concentration of 1 × 106 cells / well and simultaneously stimulated with lps ( 5 μg / ml ) for 48 hours with or without apotf ( 12 . 5 μg / ml ), peritoneal cells from balb / c mice and c57bl mice were placed in a culture at a mean concentration of 5 × 106 cells / well , and incubated for 24 hours with or without lps ( 5 μg / ml ), and with or without apotf ( 12 . 5 μg / ml ). the concentration of tnf - α was established using the elisa test with a sensitivity limit of 15 pg / ml . for the purpose of assessing the production of interleukin - 1β ( il - 1β ), cba mouse peritoneal cells were divided into two sets , placed in culture at a mean concentration of 1 , 000 , 000 cells / well and simultaneously stimulated with lps ( 5 μg / ml ) for a period of 5 or 24 hours , with or without apotf ( 12 . 5 μg / ml ). the analysis of the effects of apotf on the functional parameters of the lymphomonocytes in question demonstrated that the molecule has an pleiotropic biological immunomodulatory activity on all the cell types when used in the range of concentrations examined . in fact , apotf is effective in modifying proliferative response , cytokine secretion and nitrite production by lymphomonocytes of various origin in vitro . in particular , the data in our possession suggest that an increase in the absolute number of lymphomonocytes coincided not with a parallel increase in their functional activity , but with a reduction instead , as demonstrated by the low levels of tnf - α and il - 2 detected in the cultures considered . we assessed the effects of administering apotf in vivo in a murine model of eae in sjl mice , considered the model most closely resembling human multiple sclerosis , of which it faithfully reproduces many immunobiological , clinical and histopathological aspects . briefly , the disease was induced in female sjl mice 6 to 7 weeks old , which — after undergoing a one - week period of adaptation to the stabling conditions , involving standard laboratory conditions with free access to food and water , and controlled conditions of temperature and humidity — were inoculated subcutaneously ( s . c . ), under anaesthesia , at two different axillary lymph node draining sites with 75 μg / mouse of plp ( genemed synthesis san francisco , calif .) dissolved in saline solution and emulsified in a ratio of 1 : 1 with freund complete adjuvant ( fca ) containing 0 . 6 mg of mycobacterium tuberculosis strain h37ra . on day 0 and day 2 after immunisation , the animals were also inoculated intraperitoneally ( i . p .) with 200 ng / mouse of pertussis toxin . they were then divided by means of a randomisation process into groups and treated with i . p . apotf at doses of 0 . 1 , 1 and 2 . 5 mg / kg , and with the corresponding vehicle ( sterile pbs ) once a day starting from the 7th day after induction and up until the 30th day . the animals were checked daily by an observer unaware of the treatment , who recorded their weight and assessed their clinical disease parameters according to the following criteria : 0 = no clinically evident symptoms ; 1 = flaccid tail and / or mild rear limb weakness ; 2 = flaccid tail with moderate rear limb weakness ; 3 = severe rear limb weakness and mild front limb weakness ; 4 = rear limb paralysis with moderate front limb weakness ; 5 = tetraplegia and / or animal dying . the significant parameters for the purposes of assessing the effects of the drug were : mean cumulative score ; mortality index ; time of onset of disease ; duration of disease . fig6 shows the course of the disease recorded in two different experiments conducted using the same experimental procedures . the analysis of the effects of apotf on the clinical disease parameters in question , particularly when administered at a daily dose of 2 . 5 mg / kg starting from the 7th day after induction of the disease , and thus according to a therapeutic regimen , showed that the molecule has the capacity to favourably influence the course of the experimental allergic encephalomyelitis , reducing both the mean cumulative score and the duration of the disease in the animals in question . the treatment was also well tolerated , since there was no significant difference in the mortality rates between the groups . pharmaceutical preparations containing apotf could therefore be used for the treatment of multiple sclerosis . we evaluated the effects of administering apotf in vivo on animal models of rheumatoid arthritis , such as the arthritis induced by adjuvant in lewis rats and the arthritis induced by collagen type 2 in c57bl / 6 mice . concerning the former , microscopic joint alterations tend to become clinically evident within 14 days of induction . the severity of the disease usually increases in the first two weeks , then diminishes gradually over the course of 1 - 3 weeks afterwards . joint swelling and deformity may also persist in the longer term , particularly involving the ankle . this model of disease shares significant features with human rheumatoid arthritis : in fact , aia is an inflammatory disease immunologically mediated by t cells and macrophages . briefly , the disease was induced in male lewis rats aged between 8 and 12 weeks and weighing between 170 g and 215 g , which — after undergoing a one - week period of adaptation to the stabling conditions , involving standard laboratory conditions with free access to food and water , and controlled conditions of temperature and humidity — were inoculated intradermally ( i . d .) at the base of the tail with an emulsion comprising a total volume of 100 μl , containing 0 . 3 mg of heat - killed mycobacterium tuberculosis strain h37ra and freund incomplete adjuvant . the animals were then divided by means of a randomisation process into groups and treated according to a prophylaxis regimen six times a week , starting from the day after induction of the disease and up until 30 days after induction , with i . p . apotf at doses of 1 and 2 . 5 mg / kg , with the corresponding vehicle ( sterile pbs ) and with dexamethasone at a dose of 0 . 3 mg / kg , or according to a therapeutic regimen adopting the same experimental design but starting from the 10th day after induction and up until the 30th day . the animals were checked on alternate days by an observer unaware of the treatment , who recorded their weight and assessed their clinical disease parameters according to the following criteria : the clinical arthritis index for each animal was calculated by combining the four scores for each paw . clinical severity was also established by quantifying the weekly variation in paw volume by plethysmometry ( model 7140 ; ugo basile ). the results are shown in fig7 a , 7 b , 7 c and 7 d . the activity of apotf was subsequently evaluated on arthritis induced by collagen type 2 . an advantage of this model of arthritis over other models of arthritis , such as the one induced by adjuvant , lies in the development of an arthritis - generating response to a clearly - defined antigen ( collagen type ii ), which also enables the study of antigen - induced immunological phenomena and their selective modifications induced by pharmacological treatments . briefly , the disease was induced in male dbaj / 1 mice between 8 and 9 weeks old , which — after undergoing a one - week period of adaptation to the stabling conditions , involving standard laboratory conditions with free access to food and water , and controlled conditions of temperature and humidity — were inoculated intradermally ( i . d .) at the base of the tail with an emulsion comprising a volume of 100 μl containing 100 μg of bovine collagen type ii emulsified in freund complete adjuvant . on day 20 after immunisation , the animals received a second i . d . inoculation of 100 μg of collagen type ii dissolved in pbs for a total volume of 100 μl . they were then divided into groups using a randomisation process and treated i . p . according to a prophylactic regimen six times a week , starting from the day after induction of the disease and up until post - induction day 30 , with apotf at doses of 1 and 2 . 5 mg / kg , with the corresponding vehicle ( sterile pbs ), and with dexamethasone at a dose of 0 . 3 mg / kg , or according to a therapeutic regimen adopting the same experimental design but starting from the 22nd day after induction and continuing up until the 30th . the same disease assessment criteria were applied as described previously for adjuvant - induced arthritis . the results are shown in fig8 a , 8 b , 8 c and 8 d . the analysis of the effects of apotf on the parameters examined showed that it is effective in reducing both the clinical score for the disease and the mean volume of the paw in both the animal models of rheumatoid arthritis and with both the drug administration regimens considered , especially at a dose of 2 . 5 mg / kg . in fact , the results were similar to those obtained with the medication used as a positive control , i . e . dexamethasone . the treatment was also well tolerated , as shown by the absence of any significant differences in the animals &# 39 ; bodyweight or the number of lethal events recorded by comparison with the group treated with the vehicle . thus , pharmaceutical preparations containing apotf could be used for the treatment of human rheumatoid arthritis and in general of diseases involving joint damage with an immunoinflammatory pathogenesis . we assessed the effects of administering apotf in vivo on colitis induced by dinitrobenzene sulphonic acid ( dnbs ) in lewis rats . in this model , a single intracolonic administration of dnbs triggers the onset , within 4 days , of a pathological condition with clinical and immunohistological characteristics entirely similar to those seen in human intestinal inflammatory diseases , such as crohn &# 39 ; s disease and ulcerous recto - colitis . these include extensive damage to the colon , with areas of hyperaemia , oedema and ulceration , as well as an increase in the weight of the colon , which is microscopically characterised by the presence of necrosis , inflammation and fibrosis . as in its human counterpart , it seems that activation of the t cells and macrophages , accompanied by an overproduction of pro - inflammatory cytokines type 1 , such as tnf - α , is the main pathogenic mechanism behind the condition . briefly , the disease was induced in male wistar rats weighing between 200 and 250 g , which — after undergoing a one - week period of adaptation to the stabling conditions , involving standard laboratory conditions with free access to food and water , and controlled conditions of temperature and humidity — on day 0 , after 24 hours of fasting and under mild anaesthesia , were administered intracolonically ( i . c .) a solution containing 0 . 25 ml of 50 % ethanol with 30 mg of dnbs by means of a catheter with an external diameter of 0 . 3 mm positioned approximately 7 cm proximally to the anus . after the dnbs administration , 0 . 5 ml of air were insufflated and the catheter was removed . the animals were then kept for approximately 30 seconds in a trendelenburg position . the animals were divided into groups using a randomisation process and treated i . p . with apotf at daily doses of 2 . 5 and 5 mg / kg , with the corresponding vehicle ( sterile pbs ) and with dexamethasone at a dose of 1 mg / kg , for five consecutive days , starting from the day before induction ( days − 1 , 0 , 1 , 2 and 3 ). the animals were sacrificed on the fourth day after induction ( day 4 ) by co 2 inhalation and the segment of colon corresponding to the distally terminal 10 cm was harvested . the colon was emptied of any enteral material contained in the cavity then weighed , cut longitudinally and opened for weighing , to assess the area of damaged mucosa ( adm ), corresponding to the area of macroscopically visible necrosis , with the aid of a calibre , and to assess the microscopic damage score ( mds ) according to the following criteria : 0 = no damage ; 1 = localised hyperaemia and / or oedema ; 2 = linear ulcer & lt ; half the circumference of the colon ; 3 = linear ulcer & gt ; half the circumference of the colon ; 4 = circular ulcer & lt ; 1 cm ; 5 = circular ulcer between 1 and 2 cm long ; 6 = circular ulcer & gt ; 2 cm long . the animals &# 39 ; body weight was also recorded , both on the day of induction and on the day of death . the results of two exemplary experiments , performed using the same experimental design , are shown in fig9 a and 9b . the analysis of the effects of apotf on the parameters considered showed that , when administered at 5 mg / kg , it is effective in reducing both the area of necrosis and the microscopic damage score associated with the disease . in fact , the results were comparable , and in some cases even better than those obtained with the medication used as a positive control , i . e . dexamethasone , which only proved able to reduce the weight of the colon . for these reasons , pharmaceutical preparations containing apotf could be used for the treatment of human inflammatory intestinal diseases . we assessed the capacity of apotf to influence the onset and course of diabetes mellitus type 1 in various animal models , in spontaneous form , accelerated with the transfer of splenic cells from syngenic diseased animals , or induced by administering cyclophosphamide in nod mice , and spontaneous diabetes in bb rats . nod mice spontaneously develop a diabetes mellitus mediated by t - cell dependent autoimmune mechanisms at between 80 and 200 days of life , with an incidence of 80 % in females and 10 - 20 % in males . the clinically manifest onset of diabetes is preceded by an inflammatory infiltration of the pancreatic islets ( insulitis ), which can generally be identified on histological examination starting from 4 - 6 weeks of life . in some mouse strains it is also possible to induce an accelerated form of cell - mediated autoimmune diabetes by transferring splenic cells from diabetic nod mice to syngenic euglycaemic animals , or by means of one or more injections ( 200 - 300 mg / kg ) of cyclophosphamide administered with a two - week interval between them . to assess the effects of apotf on spontaneous diabetes in nod mice , 4 or 8 - 9 week - old female mice of said strain underwent a one - week period of adaptation to the stabling conditions , involving standard laboratory conditions with free access to food and water , and controlled conditions of temperature and humidity before the experiment ; then they were respectively treated intraperitoneally with apotf at a dose of 2 . 5 mg / kg , and with the corresponding vehicle ( sterile pbs ) for six days a week for 12 consecutive weeks , or with apotf at doses of 0 . 1 mg / kg , 1 mg / kg , 2 . 5 mg / kg , and the corresponding vehicle ( sterile pbs ), according to the same experimental design . during the study period , the mice were checked twice a week for the onset of diabetes by measuring their glycosuria : then they were confirmed as being diabetic when their fasting glycaemia levels were found higher than 12 mmol / l on two consecutive days . the animals that began the treatment at 4 weeks old were sacrificed by co 2 inhalation at the end of the treatment period and their pancreas was harvested for histological assessment . this histological examination of the pancreatic islets was conducted with reference to a sample scale according to the following criteria : at least 12 islets were examined for each mouse , while the mean histological score for each pancreas was obtained by dividing the total score by the number of islets examined . the results obtained from the means of two separate experiments are presented in fig1 a and 10b . to assess the effects of apotf on accelerated diabetes in nod mice , 5 or 13 - 14 week - old female mice of this strain underwent a one - week period of adaptation to the stabling conditions , involving standard laboratory conditions with free access to food and water , and controlled conditions of temperature and humidity before the experiment ; then diabetes was induced either by means of the intravenous transfer ( to the tail vein ) of splenic cells isolated from diabetic nod mice into euglycaemic nod mice , at a concentration of 2 . 5 × 10 6 cells / mouse , or by means of two intraperitoneal injections of cyclophosphamide at a dose of 200 mg / kg , administered 14 days apart . the onset of diabetes was identified by means of daily glycosuria measurements , diagnosing animals as diabetic when , after two positive glycosuria tests , their fasting glycaemia was found higher than 12 mmol / l on two consecutive days . the animals were assessed for glycosuria on alternate days throughout the course of the experiment . for the model of accelerated diabetes induced by cell transfer , the animals were randomised to be treated intraperitoneally ( i . p .) with rat monoclonal antibody or mouse anti - ifn - gamma ( an - 18 ) at a dose of 500 μg / mouse on alternate days ( as a positive control ), with apotf at doses of 1 mg / kg and 2 . 5 mg / kg , or with the corresponding vehicle ( sterile pbs ), starting from the seventh day after the cell transfer , six times a week for six consecutive weeks . for the accelerated diabetes induced by cyclophosphamide inoculation , the animals were randomised and treated intraperitoneally ( i . p .) with apotf at doses of 1 and 2 . 5 mg / kg , with the corresponding vehicle ( sterile pbs ) six times a week for 28 days , and with anti - ifn - γ monoclonal antibody an - 18 at a dose of 500 μg / mouse on alternate days for 28 consecutive days , according to a prophylactic regimen , or using the same molecules and the same experimental design but starting from the seventh day after cyclophosphamide inoculation and throughout the experimental period up until the 28th day . in both cases the animals were euglycaemic at the start of the therapy . the results obtained from the means of two separate experiments are presented in fig1 c , 10 d and 10 e . like nod mice , bb rats spontaneously develop insulin - dependent diabetes mellitus due to the destruction of the pancreatic islet mediated by autoreactive t cells . here again , clinically manifest disease is preceded by an inflammatory infiltration of the pancreatic islets , but diabetes develops acutely at between 60 and 120 days of life , with an incidence of 60 - 80 % in both males and females . to assess the effects of apotf on spontaneous diabetes in the bb rat , male rats of this strain aged between 37 and 46 days of life first underwent a one - week period of adaptation to the stabling conditions , involving standard laboratory conditions with free access to food and water , and controlled conditions of temperature and humidity before the experiment ; then they were treated with i . p . apotf at doses of 1 . 25 mg / kg , 2 . 5 mg / kg , 5 mg / kg , and with the corresponding vehicle ( sterile pbs ) every day for seven consecutive weeks . the onset of diabetes was assessed by measuring glycosuria and diagnosing as diabetic any animals with a fasting glycaemia found higher than 12 mmol / l on at least two consecutive days . the results obtained from the means of two separate experiments are presented in fig1 f , 10 g and 10 h . the analysis of the effects of apotf on the parameters examined showed that , especially when administered at a dose of 2 . 5 mg / kg , it is effective in reducing both the insulitis and the incidence of diabetes according to the experimental design adopted in the models of diabetes mellitus type 1 , be it spontaneous or accelerated by cell transfer or cyclophosphamide inoculation , in nod mice . in the light of the above , pharmaceutical preparations containing apotf could be used for the prevention and treatment of diabetes mellitus type 1 in humans . we evaluated the effects of administering apotf in vivo on hepatitis induced by cona in nmri mice , which represent a useful model of numerous human liver diseases with an immunoinflammatory pathogenesis . in this model , a single injection of cona suffices to develop immunomediated liver lesions . in fact , within 8 - 24 hours of cona administration , the pathological picture is characterised by clinical and histological signs of hepatitis with an increased transaminase activity in the plasma , intra - lobular inflammatory infiltration with a massive accumulation of granulocytes , and necrotic changes in the liver cells . the hepatitis induced by cona is dependent both on the cd4 + t cells and on the macrophages . briefly , the disease was induced in male nmri mice aged between 6 and 7 weeks , which — after undergoing a one - week period of adaptation to the stabling conditions , involving standard laboratory conditions with free access to food and water , and controlled conditions of temperature and humidity — on day 0 , after a 16 - hour fast , after inducing mild anaesthesia , were inoculated intravenously ( i . v .) through the tail vein with 20 mg / kg of cona dissolved in sterile pbs . they were divided into groups by means of a randomisation process and treated with i . p . apotf at doses of 0 . 1 , 1 and 2 . 5 mg / kg , and with the corresponding vehicle ( sterile pbs ) 24 hours and then 1 hour before inoculation with cona , and they were sacrificed 8 hours after inoculation with cona to collect peripheral venous blood samples . glutamate - pyruvate transaminase ( gpt ) activity in the plasma was then determined using a photometric standard , tested using the chromatic analyser . the analysis of the effects of apotf on the parameter in question showed that , administered at 2 . 5 mg / kg , it is effective in reducing the extent of liver damage associated with the administration of cona .