it is a commonly held belief that persistent hiv infection is maintained by a subset of latently infected , long - lived t and non - t cell populations 1 - 2 . potential sanctuaries for the persistent hiv infection include natural killer cells , astrocytes , microglia , b cells , dendritic cells , macrophages , and memory t cells 3 - 7 . macrophages and memory t cells are more commonly considered as the most significant stable reservoirs for persistent hiv infection because of their longevity , wide distribution , and the ability to harbor replication - components of the virus 3 - 7 . unfortunately , there is neither an established method to quantify the number of latently infected cells in hiv patients , nor is there a highly representative in vitro model that could be used to quantitatively monitor the entire process of hiv infection 8 . in addition , most studies have been preferentially focused on peripheral blood , tissue cultures and animal models , and have not looked at h&e stained sections of organs from patients with hiv . thus , the dynamics of hiv infection and the extent of contributions of each hiv reservoir to development and progression of hiv infection in different sites of human tissues remain elusive 8 . furthermore , new types of cells are continually implicated as hiv reservoirs 9 - 10 . based on results from studies in human tissue cultures , venous and cord blood , and placental tissues with immunophenotyping , image analysis , real - time pcr , and elisa assay , sundstrom et al have recently advanced a new hypothesis that human tissue mast cells are an inducible reservoir for persistent hiv infection 11 . this hypothesis postulates that unlike other hiv vulnerable cell lineages , progenitor mast cells ( prmcs ) are susceptible to infection during a limited period of their ontogeny . after infection with hiv , prmcs lose the expression of viral chemokine coreceptors and develop into long - lived , latently infected mature mast cells that are resistant to new hiv infection . in vivo recruitment of prmcs occurs in response to tissue injury , growth , remodeling , allergic reactions , or inflammation , which allow hiv - infected prmcs to spread persistent hiv infection to different tissue sites 11 . this hypothesis regarding the function of prmcs in hiv appears to be supported by some recent studies : ( 1 ) a previous study by a different group has shown that prmcs can be infected by hiv and retain virus with maturation in vitro 12 , ( 2 ) increasing studies have shown that mast cells are capable of regulating inflammation , host defense , or innate immunity , and ( 3 ) the number and functions of mast cells change significantly following hiv infection 13 - 15 . this hypothesis , however , has not been validated in different tissue sites of hiv - infected individuals . therefore , our study attempted to validate this hypothesis by statistically comparing the distribution of mast cells and p24 expressing cells in different tissue sites of hiv infected patients . paraffin - embedded tissue blocks from different anatomic sites ( lymph node , cervix , parotid glands , nasopharynx , and gi - tract ) of 10 hiv infected patients were retrieved from the files of the armed forces institute of pathology with consent from the contributors . all patients were known to be hiv - infected and none had mast cell - related disease . consecutive sections at 4 - 5um thickness were cut from each block and placed on positively - charged microscopic slides . the first and last sections from each case were subjected to h&e staining for morphological classification . a mouse monoclonal antibody against human hiv p24 capsid protein was purchased from novocastra laboratories , ltd ( newcastle , uk ) . a mouse monoclonal antibody ( clone : aa1 ) against human mast cell tryptase was purchased from dako ( giostrup , denmark ) . the corresponding secondary antibody , abc detection kits , and substrate kits were obtained from vector laboratories ( burlingame , ca ) . to assess potential co - localization ( defined as the expression of two proteins within the same cell ) of p24 and mast cell tryptase , three technical approaches were used . first , sets of two immediate adjacent sections from each case were subjected to immunostaining for p24 and mast cell tryptase , respectively , using our published protocols 16 - 17 . after immunostaining , 5 - 7 identical areas at two different sections of the same case were photographed , and the cellular distribution of these two proteins in enlarged prints was examined , to determine whether immunoreactivities to these two proteins would be co - localized within the same cells . second , a single section from each case was subjected to double immunohistochemistry for mast cell tryptase and p24 , as previously described 16 - 17 . after immunostaining , all large p24 positive cell clusters ( at least 10 p24 positive cells / per cluster ) in each case were photographed , and the distribution of these two proteins in enlarged prints was examined , to determine whether they would be co - localized within the same cells . in addition , all isolated individual p24 or mast cell tryptase positive cells in each case were photographed , and the distribution of these two proteins in enlarged prints was examined , to determine whether they would be co - localized within the same cells . third , as previous studies have well documented that a subset of macrophages is a significant reservoir for latent hiv infection and replication , and that macrophages and mast cells share a similar mechanism of in vivo recruitment 18 - 19 , two adjacent sections from each case were double immunostained for p24 plus mast cell tryptase , and p24 plus macrophage ( ncl - macro , novocastra laboratories , ltd , newcastle , uk ) , respectively . the distribution pattern of macrophages and mast cells relative to p24 positive cells were compared with special attention to p24 immunostaining within mast cells or macrophages . to assess the specificity of the immunostaining first , different negative controls were used , which included ( 1 ) the substitution of the primary antibody with the normal serum , ( 2 ) the omission of the secondary antibody from the immunostaining sequence , ( 3 ) a series dilution of the primary antibodies , and ( 4 ) the inclusion of non - hiv infected normal tissue sections from each site . second , the procedure was repeated at least twice using the same protocol and under the same condition . a given cell was considered positive , if distinct ( + + or more ) immunoreactivity was consistently seen in its cytoplasm , whereas all the negative controls were devoid of immunoreactivities . all 10 cases showed p24 ( + ) cells and scattered mast cells or clusters of mast cells . the evaluations among investigators were consistent . within lymph nodes , the p24 antibody showed a dendritic staining pattern and marked cells within follicular dendritic areas ( surface staining presumed to be trapped virus rather than productive infection ) . in two immediate adjacent sections immunostained for mast cell tryptase and p24 , respectively , mast cells and p24 expressing cells were differentially distributed . as shown in fig 1 , a vast majority of p24 expressing cells are arranged as densely packed clusters with different sizes and shapes , while a vast majority of mast cells are found scattered and individually distributed . substantially fewer mast cells are found adjacent to , compared to those at a distance from , p24 expressing cell clusters . the distribution pattern of p24 expressing and mast cells in all other tissue sites , including cervix , parotid glands , nasopharynx , and gi - tract was very similar to that in lymph nodes . in sections double immunostained for mast cell tryptase and p24 with two differently colored substrates , 455 ( 98.9% ) of the 460 large p24 expressing cell clusters encountered had no immediately surrounding mast cells ( fig 2 ) . only 5 ( 1.1% ) of 460 p24 expressing cell clusters showed the distribution of mast cells within or adjacent to p24 expressing cell clusters , whereas no distinct co - localization of these two proteins was observed ( table 1 ; fig 2 ) . similarly , no distinct co - localization was seen among over 500 isolated individual mast or p24 expressing cells examined ( table 2 ; fig 3 ) . the distribution of macrophages sharply differed from that of mast cells in two aspects : 1 ) macrophages were consistently intermixed with or adjacent to p24 expressing cells , and 2 ) p24 immunostaining were seen in the cytoplasm of a subset of macrophages ( fig 4 ) . it has been well documented that direct physical contact among different cell types is the primary mechanism for hiv infection and aids pathogenesis 21 . the spread of hiv infection from reservoirs to different tissue sites is likely to share the same mechanism for two main reasons : 1 ) in vivo recruitment of prmcs occurs in response to tissue injury , growth , remodeling , allergic reactions , or inflammation ; 2 ) several hiv susceptible tissue sites , including the lymph node , cervix , gi - tract , salivary glands , and male genital tract , normally harbor abundant prmcs 9 - 10 , 22 - 24 . although hiv infected prmcs may gradually lose the expression of viral chemokine coreceptors , the re - seeding or spread of the virus apparently requires the re - synthesis of the viral proteins and replication of the archival clone of the virus in the reservoir . the reactivation of latent hiv infection in the reservoir could be triggered by a number of factors , including t - cell activation , herpes - virus infection , or antifungal agent amphotericin b 25 - 27 . thus , it is likely that a significant number of latently infected mast cells at the stage of reactivation will express both mast and viral specific proteins , or at least , they would be physically blended within or immediately adjacent to hiv infected cells in different tissue sites of hiv patients . in contrast to our speculation , our study reveals differential distribution of mast and hiv infected cells , and a separate localization of mast cell and viral specific proteins in all or nearly all the sites and cells observed . the most likely explanation for our findings is that these mast cells are not derived from hiv infected circulating progenitors . in agreement with our speculation is the fact that previous studies have well documented that progenitor mast cells derived from undifferentiated bone marrow are susceptible to hiv infection for only a short period of time 12 . thus , the establishment and number of hiv infected mature mast cells as a hiv reservoir will be largely dependent on the active recruitment of the susceptible circulating progenitors at the initial stage of hiv infection . in our cases , the number of the susceptible circulating progenitors might be very small , or no susceptible circulating progenitors were recruited at the initial stage of hiv infection . alternatively , mast cells seen in our study might represent a latently hiv infected population that is not currently reactivated . in agreement with our speculation is that previous studies have documented that mature mast cells , unlike macrophages , no longer express a detectable level of viral chemokine coreceptors and may have become resistant to hiv infection 11 - 12 . the consistent lack of mast cells in areas surrounding p24 expressing cells , which is in sharp contrast to macrophages that are consistently intermixed with or adjacent to p24 expressing cells , suggest that mast cells may even have the capability to repel the in vivo recruitment into hiv infected sites . in either case , our findings appear to have significant scientific and clinical implications . if further studies are unable to detect viral genetic sequences in mast cells , a statement could be made that mast cells may not be a significant reservoir for persistent hiv infection . on the other hand , if integrated viral gene sequences are detectable , mast cells could be potentially used as a unique model to study latent hiv infection . since hiv reservoirs are the primary source of persistent hiv infection and drug resistance , identification of the predominant reservoirs , and the specific contribution and interactions among different reservoirs may lead to more effective interventions , and consequently improve prognosis .
a recent report postulated that the mast cell population is a significant reservoir for persistent hiv infection . 
 our study attempted to validate this hypothesis by quantitatively comparing the distribution of mast cells and cells expressing the hiv protein p24 in hiv infected patients . 
 consecutive sections of paraffin - embedded human tissues from various tissue sites were subjected to immunohistochemistry with monoclonal antibodies to mast cell tryptase , viral protein p24 , and other molecules . 
 the sub - cellular distribution of these molecules was examined , to determine whether immunoreactivities to these molecules would be co - localized within the same cells . 
 our study revealed that , in two immediate adjacent sections immunostained for mast cell tryptase and p24 , respectively , all or nearly all tryptase and p24 expressing cells were distributed at different areas . in the single section 
 double immunostained for mast cell tryptase and p24 , 5 ( 1.1% ) of 460 large p24 expressing cell clusters encountered showed a single or few mast cells within or adjacent to p24 expressing cell clusters , but no distinct co - localization of these two proteins was observed . 
 similarly , no distinct co - localization was observed in any of over 500 isolated individual mast cells and p24 expressing cells . 
 in contrast , macrophages were consistently intermixed with or adjacent to p24 expressing cells , and p24 immunostaining were seen in the cytoplasm of a subset of macrophages . 
 these findings suggest that tissue mast cells do not show evidence for active virus replication by the techniques employed .