text: the diagnostic method described herein was developed using nineteen patients exhibiting kawasaki disease (&# 34 ; kd &# 34 ; hereafter ). the patients , all children , had been admitted to three different hospitals . for the experiments which follow , &# 34 ; acute kd &# 34 ; was defined by criteria including ( i ) fever ; ( 2 ) non exudative conjunctival infection ; ( 3 ) oral pharynx changes including mucosal erythema , dry and fissured lips , &# 34 ; strawberry tongue &# 34 ;; ( 4 ) changes in extremities including erythema of palms and soles , edema of hands and feet , or , in the subacute phase of the disease , periungual desquamation ; ( 5 ) rash and ( 6 ) cervical adenopathy ( at least one node , 1 . 5 cm or more in diameter ). fever , plus four of the remaining five indications were required for a diagnosis of acute kd . in addition , the patients could not show clinical and / or laboratory evidence of other diseases known to mimic kd . blood was drawn before treatment ( ivgg ) in 14 patients with acute kd ( within 10 days of onset of fever ), and after 40 days of onset of fever in 11 patients (&# 34 ; convalescent &# 34 ; kd ). these data resulted in six paired samples from acute and convalescent stages . as controls , eight children and twelve adults served as healthy controls , and 10 subjects were studied as disease controls . of the disease controls , four were children with viral syndromes associated with fever and rash , but who did not fulfill the kd diagnostic criteria discussed supra ; one child with culture proven measles ; one child with symptomatic systemic juvenile rheumatoid arthritis , one with pauci articular jra , one child with fever and pneumococcal brain abscess , one adult with scarlet fever , and one adult with fever , rash , lymphadenopathy and jaundice . table i , reproduced infra , provides demographics . table 1__________________________________________________________________________demographic data of patients and controls acute kd conv kd healthy children healthy adults disease controls__________________________________________________________________________number 14 11 8 12 10median age 27 mo 25 mo 16 mo 33 yrs 5 yrs ( range ) ( 5 mo - 11 yrs ) ( 7 mo - 6 yrs ) ( 7 mo - 3 yrs ) ( 26 mo - 40 yrs ) ( 12 mo - 18 yrs ) sex ( male : female ) 6 : 8 6 : 5 4 : 4 10 : 2 5 : 5median days after 6 d 63 d 5 donset of fever ( range ) ( 3 d - 10 d ) ( 44 d - 153 d ) ( 1 d - 10 d ) race or ethnic groupjapanese 10 7 0 6 1mixed 2 1 0 0 0non - japanese 2 3 8 6 9__________________________________________________________________________ for patients , blood was drawn as indicated supra , and peripheral blood mononuclear cells (&# 34 ; pbmc &# 34 ;) were isolated from heparinized blood using ficoll - hypaque density gradient centrifugation , which is a well known and standard technique . these pbmc samples contain t - cells , and to increase production of mrna for the t cell receptor molecules (&# 34 ; tcrs &# 34 ; hereafter ), the t cells were stimulated for three days at 37 ° c ., in an atmosphere with 95 % humidity and 5 % co 2 , using anti - cd3 antibodies ( 20 ng / ml ). the cells were washed , and then expanded for one day in an interleukin - 2 containing medium , as described by choi et al ., j . exp . med . 172 : 981 - 4 ( 1990 ); kappler et al ., science 244 : 811 - 3 ( 1989 ). this methodology has been found to not selectively expand vβs studied herein , as compared to freshly isolated pbmc . polymerase chain reaction methodology (&# 34 ; pcr &# 34 ;) was used to estimate the proportion of t cells expressing a particular vβ gene segment . the basic methodology is described by choi et al ., proc . natl . acad . sci . u . s . a . 86 : 8941 - 4 ( 1989 ), the disclosure of which is incorporated by reference . in these pcr experiments , total rna was prepared from stimulated cells , using the acid guanidium - phenol chloroform (&# 34 ; agpc &# 34 ;) method of chomczynski et al ., anal . biochem . 162 : 156 - 9 ( 1987 ). two micrograms of total rna were used for synthesis of the first strand of cdna . in carrying out the pcr , oligonucleotide primers included a vβ specific oligomer for one of each of the 22 vβ families analyzed , as well as an oligomer from downstream cβ (&# 34 ; constant β &# 34 ;) region , as one pair . the second pair of primers included two oligomers from the cα (&# 34 ; constant α &# 34 ;) regions . the primers have been described in choi et al ., supra , and reference is made thereto for specific sequences . it must be noted that certain primers do not distinguish between family members -- i . e ., the primers for vβ5 . 2 , vβ6 . 1 and vβ8 . 1 do not distinguish among vβ5 . 2 and 5 . 3 , vβ6 . 1 , 6 . 2 and 6 . 3 , and vβ8 . 1 and 8 . 2 , respectively . amounts of cdna used for each reaction was determined individually , using from one fortieth to one two hundredth of total cdna . when pcr was used , 23 cycles of amplification were used for vβ2 , 3 , 6 . 1 , 7 , 8 . 1 and 13 . 1 , while 25 cycles were used for all others . the difference results from differences in the original mrna preparations , and was done to ensure that synthesis was proportional . in order to quantify amplified products , 32 p end - labeled 3 &# 39 ; primers (˜ 1 × 10 6 cpm each ) were added to reactions . amplified products were separated on 2 % agarose gels , dried , and analyzed with an ambis radioanalytic image system ( ambis systems , san diego ). also , input vβ rna was analyzed as compared to amplified pcr product obtained . the values showed direct correlation between ratios of pcr product and amount of mrna . results are shown in fig1 using two acute kd patients , two convalescent kd patients , and a healthy child . the binding shows a marked increase in amplified vbβ2 and vβ8 . 1 cdna in acute patients as compared to convalescent patients and the control . the vβ6 . 1 data show little difference . when total tcr vβ expression was studied , only vβ2 and vβ8 . showed significant increased expression above controls ( vβ2 : p = 0 . 005 ; vβ8 . 1 : p = 0 . 008 ). fig2 shows representative panel of analyses . in fig3 mean vβ2 / cα ration is plotted , and it shows a significantly higher value for acute kd patients as compared to controls ( mean ± sd of0 . 24 ± 12 , acute kd ; 0 . 10 ± 0 . 02 , healthy children , p = 0 . 005 using paired t test ). for out of 14 children with acute kd , the vβ2 / cα ratios were increased by more than 2 standard deviations (&# 34 ; sd &# 34 ;) above those for healthy children . a similar pattern is found for vβ8 . 1 / co ratios ( acute kd : 0 . 12 ± 0 . 05 ; healthy patients ; 0 . 06 ± 0 . 01 , p = 0 . 008 ). nine out of 14 acute kd patients had values more than 2 sd above values for healthy children . referring to fig3 it should be borne in mind that the highest prevalence of kd is seen in japan , and 13 out of 19 subjects with kd ( 68 %) were japanese . to that end , 6 japanese and 6 non - japanese healthy adults were tested to determine if ethnic differences affect tcr . fig3 shows that there is no difference , for vβ2 and vβ8 . 1 . it was also determined that there were no differences in any of the vβ elements , but these results are not shown . additional determinations were carried out to see if selective expansion is restricted to acute kd . to do this , t cells from 11 convalescent patients were tested . only two showed elevated vβ2 / cα ratios more than 2sd above normal . a significant decrease was noted , compared to acute patients . in the case of vβ8 . 1 , no convalescent patients showed elevated ratios . when other vβ / cα ratios wee studied , it was found that only vβ2 / cα and vβ8 . 1 / cα ratios showed significant decrease between acute and convalescent phases . these data were generated from the six patients who provided paired samples . four of these patients showed elevated vβ2 and vβ8 . 1 during acute kd , and significant drops ( vβ2 : p = 0 . 04 ; vβ8 . 1 : p = 0 . 02 ) between acute and convalescent stages of the illness . a second set of assays were carried out using monoclonal antibodies specific for particular vβ elements . it is important to note that monoclonal antibodies are not available for all vβ elements known ; however , the following experiments used what was available . five biotinylated monoclonal antibodies , directed against vβ5 . 1 ; vβ5 . 2 , 5 . 3 ; vβ6 . 7 , vβ8 . 1 , 8 . 2 ; and vβ12 were mixed with samples prepared as described supra , and this mixing was followed by incubation with streptavidin - pe ( 1 / 100 ), and fitc labeled anti leu 4 ( anti cd3 ) monoclonal antibodies . the resulting complexes may be presented as : this material was analyzed using standard fluorescent cell sorting analysis . forward angle and 90 degree light scatter patterns were used to gate blast cells , which can be easily distinguished from small lymphocytes . fluorescent intensity was analyzed using a cytofluorograph , and the percentage of t cell blasts bearing each tcr vβ element was expressed as a percentage of total t cells . ( t cells are cd3 + ). the results , presented in part in fig5 showed little difference in the percentage of t cells bearing vβ5 . 1 , 5 . 2 , 6 . 7 and 12 over acute kd , convalescent kd , and control subjects . on the other hand 4 of out 7 acute kd patients showed increased vβ8 . 1 levels . when vβ8 . 1 levels were followed serially in three acute kd patients as they entered convalescence , these dropped to normal levels . indeed , of the 9 convalescent patients measured none showed elevated vβ8 . 1 levels . the foregoing data show that one can diagnose a vasculitic disorder by assaying a t - cell containing sample of a patient believed to be suffering from such a disorder for levels of a particular vβ element , where abnormal levels of the vβ element as compared to values in healthy patients are associated with the disorder . comparison of the levels thus obtained with normal levels permits the investigator to make a diagnosis . &# 34 ; vasculitic disorder &# 34 ;, as this term is used herein , refers to conditions characterized by chronic or recurrent nodular lesions of subcutaneous tissue . frequently , such disorders are characterized by obliterative inflammation of arteries and veins . examples of conditions which are encompassed in this category are given supra . in determining whether or not to assay for a vasculitic disorder , the diagnostician may consider other parameters , such as those given supra which are characteristic of a particular disorder . all pathological conditions are characterized by various clinical manifestations , which are widely available and need not be repeated here . the investigator assays a t cell containing sample from the individual being tested . various materials taken from the body contain t - cells , including blood , synovial fluid , tissue homogenates , and so forth . in addition , the samples which contain t - cells may also contain soluble or free tcrs , and the invention encompasses assaying samples containing these as well . the method of determination is up to the artisan , various techniques for assaying protein or dna being known . when pcr is used , one needs primers specific for the rna of the vβ element of interest . the amino acid sequences for the vβ elements are well known , as per toyonaga et al ., ann . rev . immunol . 5 : 585 - 620 ( 1987 ), the disclosure of which is incorporated by reference . the development of primers is thus within the ability of the artisan . monoclonal antibodies to various vβ elements are available , and the number is increasing . exemplary of teachings of specific monoclonal antibodies are kappler et al ., cell 49 : 263 - 271 ( 1987 ); mak , u . s . pat . no . 4 , 923 , 799 ; choi et al ., proc . natl . acad . sci . 88 : 8357 - 8361 ( 1991 ); acha - orbea et al ., cell 54 : 163 - 273 ( 1988 ). when using monoclonal antibodies , the various types of assay well known to the artisan , including elisa , sandwich , ifa , competitive , ria , agglutination , and so forth may be used . other methods for determining vβ element levels , including the use of dna probes , either radiolabelled or labelled with non radioactive materials as per ward et al ., u . s . pat . no . 4 , 711 , 955 may be employed as well . the observation that certain vβ subtypes are associated with particular vasculitic disorders suggests a therapeutic approach . one may treat a patient with a particular vasculitic disorder by administering an effective amount of a substance leading to specific deletion of the t - cell subpopulation presenting these vβ elements . for example , monoclonal antibodies specific for a vβ2 or vβ8 . 1 element may be used , alone or in combination to treat kawasaki disease . of course , the monoclonal antibodies , or other therapeutic agent , such as polyclonal antiserum or antisense dna which hybridizes to and blocks expression of the vβ gene , must be non - toxic and pharmaceutically effective ( e . g ., must have a high enough therapeutic index ), but the particulars of choosing these , as well as establishing appropriate dosages , are well known to the skilled artisan . the terms and expressions which have been employed are used as terms of description and not of limitation , and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof , it being recognized that various modifications are possible within the scope of the invention .