Distinct T-cell responses when BCG vaccination is delayed from birth to 6 weeks of age in Ugandan infants.

BACKGROUND: In Uganda, the tuberculosis vaccine BCG is administered on the first day of life. Infants delivered at home receive BCG vaccine at their first healthcare facility visit at 6 weeks of age. Our aim was to determine the effect of this delay in BCG vaccination on the induced immune response. METHODS: We assessed CD4(+) and CD8(+) T-cell responses with a 12-hour whole-blood intracellular cytokine/cytotoxic marker assay, and with a 6-day proliferation assay. RESULTS: We enrolled 92 infants: 50 had received BCG vaccine at birth and 42 at 6 weeks of age. Birth vaccination was associated with (1) greater induction of CD4(+) and CD8(+) T cells expressing either interferon γ (IFN-γ) alone or IFN-γ together with perforin and (2) induction of proliferating cells that had greater capacity to produce IFN-γ, tumor necrosis factor α (TNF-α), and interleukin 2 together, compared with delayed vaccination. CONCLUSIONS: Distinct patterns of T-cell induction occurred when BCG vaccine was given at birth and at 6 weeks of age. We propose that this diversity might impact protection against tuberculosis. Our results differ from those of studies of delayed BCG vaccination in South Africa and the Gambia, suggesting that geographical and population heterogeneity may affect the BCG vaccine-induced T-cell response.

The RING Domain of RING Finger 12 Efficiently Builds Degradative Ubiquitin Chains.

RNF12 is a widely expressed ubiquitin E3 ligase that is required for X-chromosome inactivation, regulation of LIM-domain containing transcription factors, and TGF-ß signaling. A RING domain at the C terminus of RNF12 is important for its E3 ligase activity, and mutations in the RING domain are associated with X-linked intellectual disability. Here we have characterized ubiquitin transfer by RNF12, and show that the RING domain can bind to, and is active with, ubiquitin conjugating enzymes (E2s) that produce degradative ubiquitin chains. We report the crystal structures of RNF12 in complex with two of these E2 enzymes, as well as with an E2~Ub conjugate in a closed conformation. These structures form a basis for understanding the deleterious effect of a number of disease causing mutations. Comparison of the RNF12 structure with other monomeric RINGs suggests that a loop prior to the core RING domain has a conserved and essential role in stabilization of the active conformation of the bound E2~Ub conjugate. Together these findings provide a framework for better understanding substrate ubiquitylation by RNF12 and the impact of disease causing mutations.

Structure of the MDM2/MDMX RING domain heterodimer reveals dimerization is required for their ubiquitylation in trans.

MDM2, a ubiquitin E3-ligase of the RING family, has a key role in regulating p53 abundance. During normal non-stress conditions p53 is targeted for degradation by MDM2. MDM2 can also target itself and MDMX for degradation. MDMX is closely related to MDM2 but the RING domain of MDMX does not possess intrinsic E3-ligase activity. Instead, MDMX regulates p53 abundance by modulating the levels and activity of MDM2. Dimerization, mediated by the conserved C-terminal RING domains of both MDM2 and MDMX, is critical to this activity. Here we report the crystal structure of the MDM2/MDMX RING domain heterodimer and map residues required for functional interaction with the E2 (UbcH5b). In both MDM2 and MDMX residues C-terminal to the RING domain have a key role in dimer formation. In addition we show that these residues are part of an extended surface that is essential for ubiquitylation in trans. This study provides a molecular basis for understanding how heterodimer formation leads to stabilization of MDM2, yet degradation of p53, and suggests novel targets for therapeutic intervention.

Bim, Bad and Bmf: intrinsically unstructured BH3-only proteins that undergo a localized conformational change upon binding to prosurvival Bcl-2 targets.

All BH3-only proteins, key initiators of programmed cell death, interact tightly with multiple binding partners and have sequences of low complexity, properties that are the hallmark of intrinsically unstructured proteins (IUPs). We show, using spectroscopic methods, that the BH3-only proteins Bim, Bad and Bmf are unstructured in the absence of binding partners. Detailed sequence analyses are consistent with this observation and suggest that most BH3-only proteins are unstructured. When Bim binds and inactivates prosurvival proteins, most residues remain disordered, only the BH3 element becomes structured, and the short alpha-helical molecular recognition element can be considered to behave as a 'bead on a string'. Coupled folding and binding is typical of many IUPs that have important signaling roles, such as BH3-only proteins, as the inherent structural plasticity favors interaction with multiple targets. This understanding offers promise for the development of BH3 mimetics, as multiple modes of binding are tolerated.

Targeting of a CD8 T cell env epitope presented by HLA-B*5802 is associated with markers of HIV disease progression and lack of selection pressure.

In HIV-infected persons, certain HLA class I alleles are associated with effective control of viremia, while others are associated with rapid disease progression. Among the most divergent clinical outcomes are the relatively good prognosis in HLA-B*5801 expressing persons and poor prognosis with HLA-B*5802. These two alleles differ by only three amino acids in regions involved in HLA-peptide recognition. This study evaluated a cohort of over 1000 persons with chronic HIV clade C virus infection to determine whether clinical outcome differences associated with B*5801 (n = 93) and B*5802 ( n = 259) expression are associated with differences in HIV-1-specific CD8 (+) T cell responses. The overall breadth and magnitude of HIV-1-specific CD8(+) T cell responses were lower in persons expressing B*5802, and epitope presentation by B*5802 contributed significantly less to the overall response as compared to B*5801-restricted CD8 (+) T cells. Moreover, viral load in B*5802-positive persons was higher and CD4 cell counts lower when this allele contributed to the overall CD8 (+) T cell response, which was detected exclusively through a single epitope in Env. In addition, persons heterozygous for B*5802 compared to persons homozygous for other HLA-B alleles had significantly higher viral loads. Viral sequencing revealed strong selection pressure mediated through B*5801-restricted responses but not through B*5802. These data indicate that minor differences in HLA sequence can have a major impact on epitope recognition, and that selective targeting of Env through HLA-B*5802 is at least ineffectual if not actively adverse in the containment of viremia. These results provide experimental evidence that not all epitope-specific responses contribute to immune containment, a better understanding of which is essential to shed light on mechanisms involved in HIV disease progression.

Tissue distribution of Diablo/Smac revealed by monoclonal antibodies.

Diablo/Smac is a mammalian pro-apoptotic protein that can antagonize the inhibitor of apoptosis proteins (IAPs). We have produced monoclonal antibodies specific for Diablo and have used these to examine its tissue distribution and subcellular localization in healthy and apoptotic cells. Diablo could be detected in a wide range of mouse tissues including liver, kidney, lung, intestine, pancreas and testes by Western blot analysis. Immunohistochemical analysis found Diablo to be most abundant in the germinal cells of the testes, the parenchymal cells of the liver and the tubule cells of the kidney. In support of previous subcellular localization analysis, Diablo was present within the mitochondria of healthy cells, but released into the cytosol following the induction of apoptosis by UV.

Solution structure and mutagenesis of the caspase recruitment domain (CARD) from Apaf-1.

Activation of procaspase-9, a key component of the apoptosis mechanism, requires the interaction of its caspase recruitment domain (CARD) with the CARD in the adaptor protein Apaf-1. Using nuclear magnetic resonance spectroscopy and mutagenesis we have determined the structure of the CARD from Apaf-1 and the residues important for binding the CARD in procaspase-9. Apaf-1's CARD contains seven short alpha-helices with the core six helices arranged in an antiparallel manner. Residues in helix 2 have a central role in mediating interaction with procaspase-9 CARD. This interaction surface is distinct from that proposed based on the structure of the CARD from RAIDD, but is coincident with that of the structurally similar FADD death effector domain and the Apaf-1 CARD interface identified by crystallographic studies.

Prognosis of clinical stage I melanoma patients with positive elective regional node dissection.

We tested 12 clinical and histologic variables to see which ones best predicted death from melanoma in 66 patients with positive elective regional node dissections (clinical stage I, pathologic stage II [CSI, PSII]). Despite the presence of lymph node metastases, not all patients had poor prognoses. Patients with tumors less than or equal to 3.5 mm and a percentage of positive nodes less than or equal to 20% had a 7-year survival rate of 66%. Within this low-risk group the subset with primary lesions on the trunk or extremities (except hands and feet) had a 7-year survival rate of 76%. This compares with poor 7-year survivals of 29% and 30% observed in other defined high-risk groups. Our results confirm and extend earlier observations concerning the prognoses of CSI, PSII melanoma patients and are relevant to any ongoing and future studies concerning elective regional node dissection (ERND) or adjuvant therapy trials in melanoma.

Lentigo maligna melanoma has no better prognosis than other types of melanoma.

We studied 48 patients with lentigo maligna melanoma (LMM) and compared the clinical stage I patients with non-LMM melanoma patients (matched by site and thickness) to see if prognosis differed. There was no significant difference in mortality from melanoma between the two groups (P = .68) after a mean follow-up time of five years (67.5 months for LMM, 60.5 months for non-LMM). In addition, a Cox multivariate analysis of the entire matched group showed that only thickness was significantly associated with death from melanoma (P = .0007) while histology (LMM v non-LMM) did not make a significant contribution (P = .61). Our data suggest that after accounting for primary tumor thickness and site, LMM and non-LMM have the same prognosis and biologic behavior, in contrast to the widely held belief that LMM has a better prognosis than other forms of melanoma.

Crystal structure of the amino-terminal coiled-coil domain of the APC tumor suppressor.

Coiled coils serve as dimerization domains for a wide variety of proteins, including the medically important oligomeric tumor suppressor protein, APC. Mutations in the APC gene are associated with an inherited susceptibility to colon cancer and with approximately 75 % of sporadic colorectal tumors. To define the basis for APC pairing and to explore the anatomy of dimeric coiled coils, we determined the 2.4 A resolution X-ray crystal structure of the N-terminal dimerization domain of APC. The peptide APC-55, encompassing the heptad repeats in APC residues 2-55, primarily forms an alpha-helical, coiled-coil dimer with newly observed core packing features. Correlated asymmetric packing of four core residues in distinct, standard rotamers is associated with a small shift in the helix register. At the C terminus, the helices splay apart and interact with a symmetry-related dimer in the crystal to form a short, anti-parallel, four-helix bundle. N-terminal fraying and C-terminal splaying of the helices, as well as the asymmetry and helix register shift describe unprecedented dynamic excursions of coiled coils. The low stability of APC-55 and divergence from the expected coiled-coil fold support the suggestion that the APC dimerization domain may extend beyond the first 55 residues.

Structure of the recombinant N-terminal lobe of human lactoferrin at 2.0 A resolution.

The three-dimensional structure of the N-terminal half-molecule of human lactoferrin, LfN, prepared by recombinant DNA methods, has been determined by X-ray crystallography at 2.0 A resolution. The protein is in its iron-bound form and is deglycosylated. X-ray diffraction data were obtained by diffractometry to 3.2 A resolution and synchrotron data collection, using Weissenberg photography with imaging plates, to 1.8 A resolution. The structure was solved by molecular replacement, using the N-lobe of native diferric human lactoferrin (Lf) as search model. Restrained least squares refinement (program TNT) has resulted in a model structure with an R-factor of 0.184 for all data 34,180 (reflections) in the resolution range 8.0 to 2.0 A. The model comprises 2490 protein atoms (residues 4 to 327), 1 Fe3+, 1 CO3(2-) and 180 solvent molecules, all regarded as water. The structure of LfN is essentially the same as that of the N-lobe of intact Lf, being folded into two similar alpha/beta domains, with the Fe3+ and CO3(2-) bound in a specific site in the interdomain cleft. These details are not affected by either deglycosylation or expression in a non-native system. At the C terminus, however, the conformation of residues 321 to 333 is changed. Whereas in Lf residues 321 to 332 form a helix crossing between the domains at the back of the iron site, in LfN residues 321 to 326 have an extended conformation, forming a third interdomain beta-strand, and residues 328 to 333 appear disordered. The conformational change is attributed to the loss of stabilizing interactions from the C-lobe and is mediated by two Gly residues, at positions 321 and 323. It is further proposed that the conformational change is responsible for the more facile iron release properties of LfN, by its effect on the hinge mechanism and increased solvent exposure of residues near the back of the iron site. Other details of the polypeptide chain conformation and the binding site have also been analysed. Two cis-proline residues are found at positions 71 and 142. The bidentate binding of the CO3(2-) to the metal ion is unambiguous, and a network of hydrogen bonds in and around the binding site links the two domains. Clearly-defined amino-aromatic hydrogen bonds are found for Arg210, near the metal site, and some 31 internal water molecules have been identified, 15 of them in essentially discrete sites, and 16 in a cluster filling a cavity in the interdomain cleft.

Preliminary crystallographic studies of the amino terminal half of human lactoferrin in its iron-saturated and iron-free forms.

The amino terminal half of human lactoferrin (LfN) produced from transfected baby hamster kidney cells has been crystallized in its iron-saturated and iron-free forms. The crystals of glycosylated LfN and deglycosylated LfN are monoclinic, space group C2, with cell dimensions a = 133.0 A, b = 58.3 A, c = 58.3 A, alpha = 90.0 degrees, beta = 114.7 degrees, gamma = 90.0 degrees, and one molecule per asymmetric unit. Crystals of apo LfN have also been prepared using deglycosylated protein. These crystals are tetragonal, space group P4(1)2(1)2 (or P4(3)2(1)2), with cell dimensions of a = b = 58.4 A and c = 217.2 A and one molecule per asymmetric unit. Both the iron-saturated and the iron-free crystals are suitable for high resolution X-ray analysis.

Altered domain closure and iron binding in transferrins: the crystal structure of the Asp60Ser mutant of the amino-terminal half-molecule of human lactoferrin.

The crystal structure of a site-specific mutant of the N-terminal half-molecule of human lactoferrin, Lf(N), in which the iron ligand Asp60 has been mutated to Ser, has been determined at 2.05 A resolution in order to determine the effects of the mutation on iron binding and domain closure. Yellow monoclinic crystals of the D60S mutant, in its iron-bound form, were prepared, and have unit cell dimensions a = 110.2 A, b = 57.0 A, c = 55.2 A, beta = 97.6 degrees, space group C2, with one molecule of 333 residues in the asymmetric unit. The structure was determined by molecular replacement, using the wild-type Lf(N) as search model, and was refined by restrained least-squares methods. The final model, comprising 2451 protein atoms (from residues 2 to 315) one Fe3+ and one CO2-(3), and 107 water molecules, gives an R-factor of 0.175 for all data in the resolution range 20.0 to 2.05 A. The model conforms well with standard geometry, having root-mean-square deviations of 0.014 A and 1.2 degrees from standard bond lengths and angles. The structure of the D60S mutant deviates in two important respects from the parent Lf(N) molecule. At the mutation site the Ser side-chain neither binds to the iron atom nor makes any interdomain contact as the substituted Asp does; instead a water molecule fills the iron coordination site and participates in interdomain hydrogen bonding. The domain closure is also changed, with the D60S mutant having a more closed conformation. Consideration of crystal packing suggests that the altered domain closure is a genuine molecular property but both the iron coordination and interdomain contacts are consistent with weakened iron binding in the mutant. The implications for iron binding in transferrins generally are discussed.

Expression of activation antigens by T cells infiltrating basal cell carcinomas.

The association of T lymphocytes and dendritic cells with the stromal mononuclear cell response to basal cell carcinomas has led to speculation that cellular immunity may, in part, regulate the growth and development of this neoplasm. It has not been established, however, whether these T cells are functionally competent, or simply coincidental bystanders. We examined the immunologic phenotypes of mononuclear cells in 32 lesions of basal cell carcinoma obtained from 26 patients. The majority of infiltrating mononuclear cells were T cells that were equally distributed between the helper/inducer (Leu 3a+) and cytotoxic/suppressor (Leu 2a+) subtypes; a minority of cells were dendritic and expressed Leu 6 antigen. Virtually all T cells and dendritic cells were HLA-DR+, and many (greater than 30%) of the T cells expressed antigens consistent with stages of ongoing activation (T9, T10). TS2/7, a novel monoclonal antibody recently documented to identify activation-specific subcomponents of 210/165/130 kD glycoprotein complex present on the surface of mitogen- or alloantigen-stimulated human T cells, was also used. Greater than 50% of the T cells observed were TS2/7+. These observations provide in situ immunomorphologic evidence of stromal T cell activation in association with basal cell carcinomas, and suggest a role for active and ongoing cellular immune mechanisms as a determinant of local biological behavior of this neoplasm.

Early death from clinical stage I melanoma.

We studied 13 prognostic factors in 582 patients with clinical stage I melanoma to determine which factor or combination of factors was associated with death from melanoma within the first 24 months following diagnosis. Thirty-six patients died during this period. Only 2 deaths occurred in patients with primary tumors thinner than 1.70 mm, and only 2 patients of 189 died with tumors located on the non-BANS extremities, excluding the hands and feet. Individual factors associated with high risk for death within 2 years included level V tumors, acral location, thickness greater than or equal to 3.65 mm, histologic ulceration greater than 3 mm, nodular type, presence of microscopic satellites, greater than 6 mitoses/mm(2), positive elective node dissection, absence of lymphocyte response at the tumor base, and absence of an associated nevus histologically. Many of the preceding individual factors are highly correlated. By the use of logistic regression analysis, only one very high risk group was found: 71 percent of patients with level V tumors greater than 1.70 mm thick with histologic ulceration width greater than 3 mm located in an area other than the extremities (excluding hands and feet) had died within 2 years of diagnosis. The ability to select high-risk groups should be useful to investigators involved with the design and evaluation of adjuvant therapy studies.

Factors associated with death from melanoma from 2 to 5 years following diagnosis in clinical stage I patients.

We studied 14 prognostic factors in 428 patients with clinical stage I melanoma to determine which factor or combination of factors was associated with death from melanoma from 24 to 60 months following diagnosis. Forty-eight patients (11 percent) died during this period. All 17 patients who had visceral metastases present at 24 months died during this period. All surviving patients were followed for at least 60 months. Individual high risk factors included ulceration width (as determined by histology), level IV or V tumor, recurrence other than visceral, 6 or more mitoses per square millimeter, presence of involved nodes on elective dissection, absent or slight lymphocyte response, tumor type other than superficial spreading, location other than extremities (excluding hands and feet), microscopic satellites, thickness, sex, and wide local excision. The presence of sex as a risk factor for patients dying from 2 to 5 years following diagnosis is noteworthy because no sex difference was noted in the early death (<24 months) group. Age, presence of a nevus, and histologic regression were not significant factors. A logistic regression analysis selected a combination of the following independent factors: (1) location on extremities excluding hands and feet (favorable), (2) thickness, (3) recurrence other than visceral, (4) positive elective nodal dissection, (5) 6 or more mitoses per square millimeter, and (6) moderate to marked lymphocyte response (favorable). Twenty-five percent of patients with level IV lesions died between 24 and 60 months compared with only a 6 percent death rate within the first 24 months.

Early death from clinical stage I melanoma.

We studied 13 prognostic factors in 582 patients with clinical stage I melanoma to determine which factor or combination of factors was associated with death from melanoma within the first 24 months following diagnosis. Thirty-six patients died during this period. Only 2 deaths occurred in patients with primary tumors thinner than 1.70 mm, and only 2 patients of 189 died with tumors located on the non-BANS extremities, excluding the hands and feet. Individual factors associated with high risk for death within 2 years included level V tumors, acral location, thickness greater than or equal to 3.65 mm, histologic ulceration greater than 3 mm, nodular type, presence of microscopic satellites, greater than 6 mitoses/mm2, positive elective node dissection, absence of lymphocyte response at the tumor base, and absence of an associated nevus histologically. Many of the preceding individual factors are highly correlated. By the use of logistic regression analysis, only one very high risk group was found: 71 percent of patients with level V tumors greater than 1.70 mm thick with histologic ulceration width greater than 3 mm located in an area other than the extremities (excluding hands and feet) had died within 2 years of diagnosis. The ability to select high-risk groups should be useful to investigators involved with the design and evaluation of adjuvant therapy studies.

Factors associated with death from melanoma from 2 to 5 years following diagnosis in clinical stage I patients.

We studied 14 prognostic factors in 428 patients with clinical stage I melanoma to determine which factor or combination of factors was associated with death from melanoma from 24 to 60 months following diagnosis. Forty-eight patients (11 percent) died during this period. All 17 patients who had visceral metastases present at 24 months died during this period. All surviving patients were followed for at least 60 months. Individual high risk factors included ulceration width (as determined by histology), level IV or V tumor, recurrence other than visceral, 6 or more mitoses per square millimeter, presence of involved nodes on elective dissection, absent or slight lymphocyte response, tumor type other than superficial spreading, location other than extremities (excluding hands and feet), microscopic satellites, thickness, sex, and wide local excision. The presence of sex as a risk factor for patients dying from 2 to 5 years following diagnosis is noteworthy because no sex difference was noted in the early death (less than 24 months) group. Age, presence of a nevus, and histologic regression were not significant factors. A logistic regression analysis selected a combination of the following independent factors: (1) location on extremities excluding hands and feet (favorable), (2) thickness, (3) recurrence other than visceral, (4) positive elective nodal dissection, (5) 6 or more mitoses per square millimeter, and (6) moderate to marked lymphocyte response (favorable). Twenty-five percent of patients with level IV lesions died between 24 and 60 months compared with only a 6 percent death rate within the first 24 months.

Prognosis in Stage 1 malignant melanoma: seven-year follow-up study of splenic radiocolloid uptake as predictor of death.

In an earlier study we found that patients with clinical Stage 1 and 2 cutaneous malignant melanoma and increased splenic radiocolloid uptake had more frequent recurrence at 24 mo, compared with melanoma patients having normal liver-spleen scintigrams. This report, an 80-mo follow-up study, gives further information on 119 clinical Stage 1 patients. Fifteen of 35 patients with increased splenic uptake (42.9%) died from melanoma as opposed to only 16 of 84 (19.1%) with normal liver-spleen images (p less than 0.01). Multivariate analysis showed that augmented splenic uptake of technetium-99m sulfur colloid is a marker for adverse prognosis in patients with malignant melanoma but does not appear to be an independent variable in predicting death. In clinical Stage 1 patients, increased splenic uptake correlated significantly with pathologic stage (positive elective node biopsy) as well as thickness and mitotic rate in patients with thicker lesions. It may be that patients with thicker, pathologically aggressive tumors have an increased splenic blood flow and/or enhanced immune and reticuloendothelial response (as manifested by abnormal liver-spleen scintigram). If so, the enhanced immune response does not appear to contribute to overall survival.

A prognostic model for clinical stage I melanoma of the lower extremity. Location on foot as independent risk factor for recurrent disease.

Thirteen variables were studied to determine their usefulness in predicting recurrent disease in 158 patients with stage I melanoma of the lower extremity. A Cox proportional hazards analysis demonstrated that three variables were independent risk factors for recurrent disease in these patients: (1) thickness, in millimeters, of the primary tumor (P = 0.000009), (2) primary tumor location on the foot (P = 0.0003), and (3) the number of mitoses/mm2 (P = 0.0244). Life-table analyses of patient subgroups defined by different combinations of these three variables demonstrated that thick (greater than or equal to 3.0 mm) melanomas of the foot were associated with recurrent disease much more frequently than tumors of similar thickness located on the thigh or calf. These data provide guidelines that can be used to evaluate results of surgical and/or adjuvant therapy studies for patients with melanoma of the lower extremity.