Faculty Bibliography

A sensitive, rapid, and quantitative ELISA for p24 is described, which can be used as the read-out to test for HIV-1 neutralization in the syncytium-forming microassay and can replace the counting of syncytia under the microscope. This assay can be used reliably for divergent strains of HIV-1, including those that do not induce syncytia. The new read-out permits evaluation of neutralization at 3 days rather than 5 days. Using this assay, the neutralizing activity of several new and previously described human monoclonal antibodies against HIV-1 was characterized

A genomic library for Plasmodium vivax was constructed in lambda gt11 and immunologically screened with pooled serum samples from vivax patients. Six seroreactive clones were isolated, and one clone, denoted PV9, was studied further. This clone has an unusual base composition (65% G + C), does not share any homology with P. falciparum, and codes for an entirely new antigenic determinant. Antibodies (immunoglobulin G type) against the PV9-encoded polypeptide were produced in all vivax patients older than 15 years. This seroreactivity was lower among patients younger than 15 years (53%). The antigenic epitope(s) of the PV9-encoded polypeptide was recognized at a similar rate by serum samples from P. vivax patients who were living 350 to 973 km apart. Fifty percent of uninfected Indian adults were also seropositive, whereas all European and American (United States) sera tested were negative, suggesting that anti-PV9 antibodies persist after infection. The seroreactivity pattern of this antigen is similar to that of the immunity developed in malaria after repeated infections

Pooled polyvalent sera from lepromatous leprosy patients were used to screen a lambda gt11 recombinant DNA expression library of Mycobacterium leprae in order to identify the relevant antigens recognized by the human immune response. Of the 300,000 phages screened, 4 clones were identified that coded for fusion proteins of the same molecular mass. The fusion protein from clone LSR2 was tested for immunoreactivity in assays using peripheral blood cells and sera from 11 laboratory personnel and 105 patients across the leprosy spectrum. LSR2 protein appears to be predominantly a T-cell antigen. It evokes similar lymphoproliferative responses as the native bacillus both at the individual level and in the leprosy spectrum as a whole. Though only 50% of patient sera with anti-M. leprae antibodies reacted with the fusion protein, the pattern of reactivity in the antibody responses was also similar for the various clinical types. The coding regions of clones LSR1 and LSR2 are identical. They show no homology with sequences stored in data banks and encode a protein of 89 amino acids with a calculated molecular mass of approximately 10 kDa

We have analyzed the nature and kinetics of a delayed, cell-mediated immune response to a purified protein derivative of tuberculin (PPD) in the skin of 154 naturally sensitized patients with lepromatous leprosy. After the intradermal injection of 5 U of PPD, biopsies were taken at 1-21 d and studied for the composition, extent, persistence, and organization of the emigratory cell response by light and electron microscopy. Induration of positive sites occurred promptly, reached a maximum diameter at 4 d, displayed a major extravasatory element, and was evident for as long as 21 d. The cellularity of the site exhibited a biphasic course, reached a maximum at 7 d, involved as much as 70% of the dermis and millions of new cells, and was elevated threefold above preinjection levels at 21 d. The emigratory cells were limited to T cells and circulating monocytes. T cells were more evident as they entered a preexisting lepromatous lesion containing parasitized macrophages and only occasional T cells many of the CD8+ phenotype. The predominant emigratory T cell was CD4+ although CD8+ cells were in evidence. The CD4/CD8 ratio of the lesions started at less than unity and in two distinct steps reached levels as high as 5:1. In most sites CD4+ cells were in the majority at 21 d. A well-defined granulomatous response with epithelioid and giant cells was apparent at 4 d, reached a maximum at 7 d, and involved all PPD sites at this time point. The generation of these differentiated mononuclear phagocytes from newly emigrated monocytes was never observed in the underlying lepromatous lesion but is a constant feature of the tuberculoid leprosy response. Epidermal thickening and keratinocyte proliferation, sequellae of the dermal reaction, reached a maximum at 7 d and gradually resolved by 3 wk. A constant feature of the PPD response was the extensive destruction of preexisting macrophages containing Mycobacterium leprae bacilli or their products. This was associated with the presence of and intimate contact with highly polarized lymphoid cells of unknown phenotype. Cell destruction did not involve other elements of the dermis and spared parasitized Schwann cells. Newly emigrated T cells and monocytes were never seen within the perineural sheath in contact with neural elements. It appears that a single antigenic stimulus leads to a very long-term, defined series of events with distinct temporal patterns. It includes waves of emigratory T cells, the maturation and organization of monocytes, the generation of killer cells, and the extensive destruction of parasitized macrophages.(ABSTRACT TRUNCATED AT 400 WORDS)

Significant epidermal changes were observed in lesions of leprosy patients undergoing type 1 (reversal) and type 2 (erythema nodosum leprosum, ENL) reactions. Using indirect immunofluorescence and frozen sections stained with the appropriate monoclonal antibodies, an increase in epidermal cell layers, the presence of Ia on keratinocytes, an increase in Langerhans' cell numbers, and scattered T cells within the epidermis were seen in both types of reactions. Although borderline tuberculoid patients with type 1 reactions showed the consistent presence of Ia on all keratinocytes, lepromatous patients undergoing ENL reactions showed only a patchy distribution. Taken together, these studies indicate that local T-cell activation leading to the production of terminal lymphokine, such as interferon-gamma, with subsequent induction of Ia on epidermal cells may be an important event in reactional leprosy states. It is of interest that the hitherto considered 'anergic' lepromatous patients should recover temporary T-cell reactivity during the natural course of the disease

Nineteen each of paucibacillary borderline tuberculoid (BT) and multibacillary borderline borderline (BB)/borderline lepromatous (BL) leprosy patients undergoing type 1 reactions were compared with nonreactional stable patients of the appropriate leprosy type. In the BT reactional group, both phytohemagglutinin-induced and, more importantly, antigen-induced lymphoproliferation was reduced in 80%-90% of the patients. On the other hand, leukocyte migration inhibition was reduced in 40% and remained unchanged in the others. Suppressor-cell activity as evaluated by a costimulant assay was also reduced in a majority of the reactional BT individuals. In contrast, the bacilliferous BB and BL patients in reaction showed significant general improvement in leukocyte migration inhibition (p less than 0.001) and antigen-induced lymphoproliferation (p less than 0.05) as compared to the expected hyporesponsive/anergic uncomplicated BB-BL patients. Suppressor-cell activity also recovered during the reactional phase. However, no significant differences were observed in either of the reactional or stable leprosy types in the numbers of total T cells (OKT3+) and their subsets as defined by OKT4+ (helper/inducer) and OKT8+ (suppressor/cytotoxic) functional phenotypes. Moreover, during type 1 reactions the 48-hr delayed-type hypersensitivity (DTH) responses after intradermal injection of Mycobacterium leprae antigens continued to reflect the background leprosy type rather than the functional perturbations in the circulating T cells. Only a marginal increase in DTH was observed in some BT reactional individuals. No consistent pattern in the above in vitro T-cell-related responses was discernable in the same individuals 4-6 months after subsidence of reactions. The clinical entity of type 1 reactions encompassing paucibacillary and multibacillary leprosy shows a heterogeneity/dichotomy in T-cell responses which may reflect different immunological mechanisms underlying the reactional state

The epidermal changes that occur in human cutaneous immune responses have been investigated in the tuberculin reaction and in the lesions of tuberculoid and lepromatous leprosy and cutaneous leishmaniasis. In each situation, there was a dermal accumulation of monocytes and T cells, and the epidermis exhibited thickening. In the tuberculin response, the thickness of the epidermis sometimes doubled in 48-72 hr, and this was attributed to increases in both size and number of keratinocytes. In addition, the phenotype of the keratinocytes changed from Ia- to Ia+. Similar changes in keratinocyte Ia-antigen expression occurred in the epidermis overlying untreated tuberculoid leprosy and cutaneous leishmaniasis lesions, but not in lepromatous leprosy. We suggest that one or more epidermal growth factors may be generated in the course of a delayed immune reaction in the dermis