in one embodiment , the present invention is a method of increasing thymic output in hiv - negative patients . in a preferred embodiment , the method comprises the step of administering an antiretroviral therapy ( art ) to an hiv - negative patient and observing an increase in thymic output . by “ thymic output ” we mean an increase in naïve t - cells , preferably as measured by phenotypic markers and tcr recombination excision circles ( trecs ), specifically signal joint ( sj ) trecs . the example below is a disclosure of one preferred embodiment of the present invention . the example analyzes trec levels in forty six volunteers , ages 22 to 95 years old . eight volunteers responded to art therapy with increases in trec numbers from base line to day 21 , with a median increase of trecs of 1 . 2 copies trec / ccr5 . sub - group analysis demonstrated an age related association . the average increase in trec levels for subjects less than 40 years was 1 . 2 trec / ccr5 copies , for subjects 40 - 65 years was 0 . 80 trec / ccr5 copies and the average change in trec levels for subjects greater than 65 years was 0 . 89 trec / ccr5 copies after art . therefore , the method of the present invention is expected to be more clinically useful to younger patients ( less than 40 years old ) but is expected to be clinically useful for some patients over 40 years old . the method of the present invention will be particularly advantageous with hiv - negative patients in need of increased thymic output . preferably , these patients would be characterized as follows : patients who would be predicted to have impaired thymic output would include elderly , chronically ill , patients who have received or are receiving immunosuppressive medications including but not limited to chronic steroid administration , immunosuppressives related to organ transplantation , having received a bone marrow transplant or chemotherapy , certain surgical procedures such as thymectomy or splenectomy . in another preferred version of the present invention , a patient has idiopathic cd4 lymphocytopenia . in one embodiment , the present invention is a method of increasing thymic output in hiv - negative patients . as described above , one would first identify a patient in need of thymic output increase . one would then supply antiretroviral therapy to the hiv - negative patient . in a preferred embodiment , we envision the use of nelfinavir mesylate , viracept , as the antiretroviral therapy . however , we envision that the present invention will be useful for any hiv protease inhibitor , such as agenerase ( amprenavir ), aptivus ( tipranavir ), crixivan ( indinavir ), invirase ( saquinavir ), kaletra ( lopinavir & amp ; ritonavir ), lexiva ( fosamprenavir ), norvir ( ritonavir ), prezista ( darunavir ), and reyataz ( atazanavir ), as well as viracept ( nelfinavir ). a preferable dose of nelvinavir is 1250 mg orally , twice a day for 21 days . we envision that one could modify this dosage and still obtain good results . for example , a dose of between 1000 - 2000 mg orally , twice a day for between 15 and 25 days is another preferred embodiment of the present invention . in general , one would supply the art in the way recommended by manufacturers of the protease inhibitors . one would wish to take a biological sample from the patient to determine if the treatment is successful . the examples below demonstrate a determination at day 3 , day 5 , day 7 , day 14 and day 21 . one may also wish to obtain a sample at day 28 . if the treatment is not producing the desired result by day 28 , we believe that the therapy will not be successful . in one embodiment , treatment of patients in the present invention will be as follows : blood will be sampled before therapy ( time 0 ), preferably at day 3 , day 5 , day 7 , day 14 , and day 21 . trec analysis : dna will be isolated from peripheral blood lymphocytes ( pbl ) using an easy dna kit ( invitrogen ) and assessed for signal joint ( sj ) trec content relative to genomic ccr5 copies by real time pcr in a spectroflourometric thermal cycler ( abi prism 7700 , pe applied biosystems ). sjtrec reactions typically contain 25 pmol primers ( forward cacatccctttcmccatgct [ seq id no : 1 ]), reverse gccagctgcagggtttagg [ seq id no : 2 ]) 125 nm taqman probe ( fam - acacctctggtttttgtaaaggtgcccact - tamra [ seq id no : 3 ), 1x taqman universal pcr master mix ( pe applied biosystems ), and 60 ng of template dna in a total volume of 50 ul . ccr5 reactions will typically contain 25 pmol primers ( forward gtgtcmgtccaatctatgacatcm [ seq id no : 4 ], reverse gcctgcgatttgcttcaca [ seq id no : 5 ]), 125 nm taqman probe ( fam - tattatacatcggagccctgccaaaaaatca - tamra [ seq id no : 6 ]), 1x taqman universal pcr master mix , and 60 ng of template dna in a total volume of 50 ul . thermal cycling conditions will typically consist of 2 minute incubation at 50 ° c . and an initial denaturation at 95 ° c . for 10 minutes , followed by 40 cycles of 95 ° c . for 10 minutes and 60 ° c . for 1 minute . the genomic ccr5 copies are determined by real time pcr . these are used as a standard to reflect the number of pbls are being evaluated . ( see procedure in examples .) for each run , standard curves will be generated , typically with 1 - 100 , 000 copies of human sjtrec plasmid ( see d . douek , et al ., nature dec . 17 , 1998 ; 396 ( 6712 ): 690 - 695 , incorporated by reference ) or human ccr5 plasmid ( cloned by rt - pcr into pci ) in order to calculate copies of trec versus ccr5 . trec values will be expressed as trec copy number per two copies of ccr5 or trec / peripheral blood leukocytes ( pbl ). preferably , success of our treatment can be monitored in the following ways : a ) one might determine trec number , immunophenotype number , or immune function by laboratory assay such as elispot before and after therapy . in a successful result , one would expect to see increased trec number , increased naïve t - cell count or increased elispot response . one would expect to see the trec measurement increase at least 1 . 2 - 3 fold , preferably at least 2 - 3 fold . b ) one might measure immune response to vaccine as assessed by antibody level and / or functional assays such as elispot assays . in a successful outcome , one would expect to see increased antibody level and increased elispot response . c ) one might measure disease incidence in patients who have received the treatment . for example , in patients receiving influenza vaccine with and without nelfinavir one would compare influenza incidence and all causes of mortality in patients receiving nelfinavir versus placebo . d ) improved thymic output may beneficially impact disease outcome from certain malignancies . thus , we envision treating patients with select malignancies ( included , but not limited to , melanoma , lymphoma , leukemia , etc .) with protease inhibitors such as nelfinavir . evaluating outcome of treatment would be compared by evaluating disease - free survival and mortality between patients receiving nelfinavir in addition to standard medical practice to patients receiving placebo in addition to standard medical practice . although patients treated with hiv protease inhibitor ( pi ) containing regimens manifest increases in naïve t - cell number , it is unclear whether this is due to reduction in viral replication or a direct drug effect . we questioned whether nelfinavir mono - therapy directly impacted naïve t - cell number in hiv - negative individuals . hiv - negative volunteers received nelfinavir 1250 bid for 3 weeks , and t - cell receptor recombination excision circles ( trec ) content in peripheral blood was assessed at baseline , during , and after 21 days of nelfinavir therapy in 15 subjects . trec copies / copies ccr5 increased following nelfinavir mono - therapy in 8 patients ( p & lt ; 0 . 02 ), and did not change in 7 patients ( p = ns ). those patients who responded were younger than those who did not with a median age of 55 years for responders and 71 years for non - responders ( p & lt ; 0 . 03 ). the increase in trec was most pronounced in those patients less than 40 years old ( p & lt ; 0 . 01 ). moreover , the patients who did not increase trec levels were more likely to have suffered a medical illness previously shown to reduce thymic function . in hiv - negative patients , mono - therapy with the hiv pi nelfinavir for 21 days increases trec positive naïve t - cell number particularly in individuals who are healthy and / or young . following approval by the mayo institutional review board , peripheral blood was sampled from 46 healthy blood donors older than 18 years of age for baseline trec analysis . for the separate nelfinavir trial , 15 hiv - negative individuals with no active illness older than 18 years of age were given nelfinavir , 1250 mg orally , twice a day for 21 days . blood for trec analysis was obtained before therapy and on days 3 , 5 , 7 , 14 , and 21 after starting therapy . at each blood draw , side - effects were monitored and recorded from each subject . severity of diarrhea was graded as : grade 0 — none ; grade 1 — increase of 4 stools / day over pretreatment ; grade 2 — increase of 4 - 6 stools / day , or nocturnal stools ; grade 3 — increase of 7 stools / day or incontinence , or dehydration episode requiring parenteral support ; grade 4 — episode of hemodynamic collapse requiring intensive care . trec analysis : dna was isolated from peripheral blood mononuclear cells ( pbmc ) using an qiaamp blood mini dna kit ( qiagen ) and assessed for signal joint ( sj ) trec content relative to genomic ccr5 copies by real - time pcr in a spectroflourometric thermal cycler ( abi prism 7700 or 7900 , pe applied biosystems ). sjtrec reactions contained 12 . 5 pmol primers ( forward cacatccctttcaaccatgct [ seq id no : 1 ]), reverse gccagctgcagggtttagg [ seq id no : 2 ]) 3 pmoles taqman probe ( fam - acacctctggtttttgtaaaggtgcccact - tamra [ seq id no : 3 ]), 1 × taqman universal pcr master mix ( pe applied biosystems ), and 50 to 400 ng of template dna in a total volume of 25 μl . ccr5 reactions contained 12 . 5 pmol primers ( forward gtgtcmgtccmtctatgacatcaa [ seq id no : 4 ], and reverse gcctgcgatttgcttcaca [ seq id no : 5 ]), 3 pmoles taqman probe ( fam - tattatacatcggagccctgccaaaaaatca - tamra [ seq id no : 6 ]), 1x taqman universal pcr master mix , and 50 - 400 ng of template of genomic dna in a total volume of 25 μl . thermal cycling conditions consisted of a 2 - minute incubation at 50 ° c . and an initial denaturation / activation step at 95 ° c . for 10 minutes , followed by 40 cycles of 95 ° c . for 15 seconds and 60 ° c . for 1 minute in a microplate format . for each plate of reactions , standard curves were generated with 1 - 100 , 000 copies of human sjtrec plasmid ( provided by dr . d . douek , nih ) and human ccr5 plasmid ( cloned by rt - pcr into pci ) in order to calculate copies of trec versus ccr5 . statistical analysis : experiments from every figure were performed in duplicate and repeated at least twice . all measurements are presented as means and standard deviations with statistical comparisons made between baseline and day 21 trec / ccr5 measurements using the student &# 39 ; s t test for paired observations . for fig2 , change in baseline to day 21 trec / ccr5 levels were stratified according to subjects age with 2 subjects & lt ; 40 , 2 subjects 40 - 65 , and 11 subjects & gt ; 65 years old . trec levels were analyzed in 46 volunteers , ages 22 - 95 years old . consistent with previous findings , 20 trec level decreased with advancing subject age ( r 2 = 0 . 6802 ) ( fig1 ). fifteen hiv - negative adults , ages 22 - 95 , were treated with nelfinavir , 1250 mg orally , twice a day for 21 days . nelfinavir mono - therapy was clinically well tolerated ; six ( 40 %) of the subjects experienced grade 1 or 2 diarrhea , and all but one subject completed the 21 days of therapy . compliance was good in all patients , as assessed by self - reporting and pill counts at the time of each blood draw . in order to establish if nelfinavir mono - therapy increased naïve t - cells after 21 days of treatment in adult subjects , a paired analysis of change in trec levels from baseline to days 3 , 5 , 7 , 14 , and 21 were determined . in our previous report , trec / ccr5 levels on t - cells did not alter over 21 days in an untreated control group of hiv - negative adults . 5 no significant change in trec levels occurred below baseline ( day 0 ) and days 3 , 5 , 7 , or 14 . after 21 days of therapy , although there was no change between baseline and day 21 trec / ccr5 levels , when the entire cohort was analyzed , it was apparent that some subjects responded to nelfinavir therapy with increasing trec / ccr5 copy levels . since no patient in our control group had any increase in trec levels over a 3 week period , we analyzed volunteers who increased trec / ccr5 levels separately . 5 eight volunteers ( 53 % of total ) responded to nelfinavir therapy with increases in trec numbers from baseline to day 21 , with a median increase in trecs of 1 . 2 copies trec / ccr5 ( p & lt ; 0 . 02 ). conversely , 7 volunteers ( 47 % of total ) did not increase trec numbers following 21 days of nelfinavir ( fig2 ). although only half of the subjects increased trec / ccr5 levels after nelfinavir therapy , sub - group analysis demonstrated an age - related association . the average increase in trec levels for subjects less than 40 was 1 . 2 trec / ccr5 copies , for subjects 40 - 65 was 0 . 80 trec / ccr5 copies and the average change in trec levels for subjects greater than 65 was 0 . 89 trec / ccr5 copies , after nelfinavir therapy . the median age of the responders was 55 years ( range 23 - 90 ), whereas , the mean age of the non - responders was 71 years ( range 37 - 88 ) ( p & lt ; 0 . 03 ), suggesting that age , and consequently , thymic reserve might determine thymic response to nelfinavir . our results confirm that the normal process of aging results in impaired thymopoiesis ( fig1 ) that may predispose to infections and cancers . 20 - 23 moreover , aging associated impairments in thymopoiesis , and / or thymic reserve appear to influence thymic response to nelfinavir therapy . ( fig2 ) younger compared to older individuals responded more robustly to nelfinavir , consistent with observations that aging is associated with reduced thymic stroma , size , function and output . 24 the overlap in ages between nelfinavir responders and non - responders is likely reflective of aging independent causes of early thymic atrophy , including puberty , pregnancy , stress , exercise and trauma , as well as disease states including cancers , diabetes , and parkinson &# 39 ; s disease ( reviewed in 24 ). of note , 5 of the 7 non - responders had prior or chronic illness that may have decreased their thymic reserve , including prostate cancer , non - small cell lung cancer , diabetes mellitus , and parkinson &# 39 ; s disease . 25 in contrast , only one of eight of the responders had a similar illness ( table 1 ). increased understanding of thymic biology has led to cytokine and hormone - based approaches to reversing thymic involution in animal models using agents such as il - 7 , 26 gh , 27 igf - 1 . 28 recognizing that hiv pi may also impact thymic function , offers new insights into potential therapies aimed at restoring thymic function in vivo . moreover , the changes observed in this study confirm and highlight the direct immunologic effects of hiv pi therapy . 4 . de rossi a , walker a s , klein n , de formi d , king d , gibb d m . increased thymic output after initiation of antiretroviral therapy in human immunodeficiency virus type 1 - infected children in the paediatric european network for treatment of aids ( penta ) 5 trial . j infect dis . aug . 1 , 2002 ; 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a method of increasing thymic output in an hiv - negative patient , comprising the steps of identifying a hiv - negative patient who is in need of increased thymic output and supplying an effective amount of antiretroviral agent to the patient such that the patient has an increase in naïve t - cells .