Rockefeller University Hospital

Passive transfer of broadly neutralizing HIV antibodies can prevent infection, which suggests that vaccines that elicit such antibodies would be protective. Thus far, however, few broadly neutralizing HIV antibodies that occur naturally have been characterized. To determine whether these antibodies are part of a larger group of related molecules, we cloned 576 new HIV antibodies from four unrelated individuals. All four individuals produced expanded clones of potent broadly neutralizing CD4-binding-site antibodies that mimic binding to CD4. Despite extensive hypermutation, the new antibodies shared a consensus sequence of 68 immunoglobulin H (IgH) chain amino acids and arise independently from two related IgH genes. Comparison of the crystal structure of one of the antibodies to the broadly neutralizing antibody VRC01 revealed conservation of the contacts to the HIV spike.

and RBD epitopes on SARS-CoV-2 spike. Moreover, a 3.4 Å cryo-electron microscopy (cryo-EM) structure of a neutralizing monoclonal Fab-spike complex revealed an epitope that blocks ACE2 receptor binding. Modeling based on these structures suggested different potentials for inter-spike crosslinking by IgGs on viruses, and characterized IgGs would not be affected by identified SARS-CoV-2 spike mutations. Overall, our studies structurally define a recurrent anti-SARS-CoV-2 antibody class derived from VH3-53/VH3-66 and similarity to a SARS-CoV VH3-30 antibody, providing criteria for evaluating vaccine-elicited antibodies.

Psoriasis is a type I-deviated disease characterized by the presence of interferon (IFN)-gamma and multiple IFN-related inflammatory genes in lesions. Because interleukin (IL)-23 is now recognized to play a role in the recruitment of inflammatory cells in a T helper cell (Th)1-mediated disease, we examined psoriasis skin lesions for production of this newly described cytokine. IL-23 is composed of two subunits: a unique p19 subunit and a p40 subunit shared with IL-12. We found a reliable increase in p19 mRNA by quantitative reverse transcription polymerase chain reaction in lesional skin compared with nonlesional skin (22.3-fold increase; P = 0.001). The p40 subunit, shared by IL-12 and IL-23, increased by 11.6-fold compared with nonlesional skin (P = 0.003), but the IL-12 p35 subunit was not increased in lesional skin. IL-23 was expressed mainly by dermal cells and increased p40 immunoreactivity was visualized in large dermal cells in the lesions. Cell isolation experiments from psoriatic tissue showed strong expression of p19 mRNA in cells expressing monocyte (CD14+ CD11c+ CD83-) and mature dendritic cell (DC) markers (CD14- CD11c+ CD83+), whereas in culture, the mRNAs for p40 and p19 were strongly up-regulated in stimulated monocytes and monocyte-derived DCs, persisting in the latter for much longer periods than IL-12. Our data suggest that IL-23 is playing a more dominant role than IL-12 in psoriasis, a Th1 type of human inflammatory disease.

Summary It is established that M protein is the cellular component of group A streptococci responsible for the production of type-specific antibodies, which are the basis of type-specific immunity in man. Strains freshly isolated from acute infections, so far as is known at present, always contain M antigen. Although many cultures containing M protein are avirulent for mice, all highly mouse-virulent group A strains contain M antigen. The amount of M protein in some cultures can be increased by serial mouse passage, and the virulence of such strains is usually increased at the same time. It is possible that the hyaluronic acid capsule in addition to M protein may be a factor in virulence, but its importance has not been clearly defined. M protein probably enhances virulence by impeding phagocytosis, and the capsule may contribute to virulence by a mechanism not yet fully elucidated. So far no completely purified preparations of M protein have been obtained, but progress in this direction is being made. Once this is achieved, it may be possible to define the properties of this important antigen more accurately.

T cells recognize peptides that are bound to MHC molecules on the surface of different types of antigen-presenting cells (APC). Antigen presentation most often is studied using T cells that have undergone priming in situ, or cell lines that have been chronically stimulated in vitro. The use of primed cells provides sufficient numbers of antigen-reactive lymphocytes for experimental study. A more complete understanding of immunogenicity, however, requires that one develop systems for studying the onset of a T cell response from unprimed lymphocytes, especially in situ. Here it is shown that mouse T cells can be reliably primed in situ using dendritic cells as APC. The dendritic cells were isolated from spleen, pulsed with protein antigens, and then administered to naive mice. Antigen-responsive T cells developed in the draining lymphoid tissue, and these T cells only recognized protein when presented on cells bearing the same MHC products as the original priming dendritic cells. In contrast, little or no priming was seen if antigen-pulsed spleen cells or peritoneal cells were injected. Since very small amounts of the foreign protein were visualized within endocytic vacuoles of antigen-pulsed dendritic cells, it is suggested that dendritic cells have a small but relevant vacuolar system for presenting antigens over a several day period in situ.

Both type I and type II diabetes are powerful and independent risk factors for coronary artery disease (CAD), stroke, and peripheral arterial disease. Atherosclerosis accounts for virtually 80% of all deaths among diabetic patients. Prolonged exposure to hyperglycemia is now recognized a major factor in the pathogenesis of atherosclerosis in diabetes. Hyperglycemia induces a large number of alterations at the cellular level of vascular tissue that potentially accelerate the atherosclerotic process. Animal and human studies have elucidated three major mechanisms that encompass most of the pathological alterations observed in the diabetic vasculature: 1) Nonenzymatic glycosylation of proteins and lipids which can interfere with their normal function by disrupting molecular conformation, alter enzymatic activity, reduce degradative capacity, and interfere with receptor recognition. In addition, glycosylated proteins interact with a specific receptor present on all cells relevant to the atherosclerotic process, including monocyte-derived macrophages, endothelial cells, and smooth muscle cells. The interaction of glycosylated proteins with their receptor results in the induction of oxidative stress and proinflammatory responses 2) oxidative stress 3) protein kinase C (PKC) activation with subsequent alteration in growth factor expression. Importantly, these mechanisms may be interrelated. For example, hyperglycemia-induced oxidative stress promotes both the formation of advanced glycosylation end products and PKC activation.

SummaryNormal human urine contains a highly active inhibitor of viral hemagglutination, effective against PR8, Lee, FM1, swine, mumps, NDV, and GDVII viruses but inactive against PVM. The inhibitor concentration may approach 2 mg%. Considerable amounts of purified material, active at 0.0001 μg/cc, can be obtained readily. The activity of the inhibitor as it occurs in urine is inversely related to the salt concentration; it should be emphasized that in NaCl solutions of moderate strength it precipitates.

Nonsteroidal antiinflammatory drugs (NSAIDs), have cancer preventive and tumor regressive effects in the human colon. They lower the incidence of and mortality from colorectal cancer and sulindac reduces the number and size of polyps in patients with familial adenomatous polyposis. We studied the effect of sulindac, and its metabolite sulindac sulfide, on the proliferation of HT-29 colon adenocarcinoma cells. Both compounds reduced the proliferation rate of these cells, changed their morphology, and caused them to accumulate in the G0/G1 phase of the cell cycle. These responses were time- and concentration-dependent and reversible. In addition, these compounds reduced the level and activity of several cyclin-dependent kinases (cdks), which regulate cell cycle progression. Sulindac and sulindac sulfide also induced apoptosis in these cells at concentrations that affected their proliferation, morphology, and cell cycle phase distribution. Sulindac sulfide was approximately sixfold more potent than sulindac in inducing these cellular responses. Our results indicate that inhibition of cell cycle progression and induction of apoptotic cell death contribute to the anti-proliferative effects of sulindac and sulindac sulfide in HT-29 cells. These findings may be relevant to the cancer preventive and tumor regressive effects of these compounds in humans.

Heme oxygenase (HO) is the rate-limiting enzyme in heme catabolism and its activity is induced by many agents, including its substrate heme, heavy metals, UV radiation, and other injurious oxidant conditions. We examined the presence of several regulatory elements in the promoter region of the human HO-1 gene which could possibly account for its induction in response to diverse agents or influences. Heme treatment increased both HO activity and HO-1 mRNA in the human erythroleukemic cell line K562. Electrophoretic mobility-shift assays of nuclear protein extracts from heme-treated and control cells with specific oligonucleotide probes containing binding sites for known transcription factors, including AP-1, AP-2, Sp1, NF-kappa B, CTF/NF1, TFIID, OKT1, and CREB, and oligonucleotides containing serum-, metal-, and glucocorticoid-responsive elements demonstrated a specific and marked increase in the NF-kappa B and AP-2 transcription factors and, to a lesser extent, an increase in AP-1. No significant increase in other transcription factors over the control, untreated cells was observed. DNase I footprint assays using purified transcription factors revealed the presence of NF-kappa B and AP-2 binding sites in the proximal part of the promoter region of the human HO-1 gene. Moreover, nucleotide sequence analysis of the HO-1 promoter region showed that the protected regions encompassed NF-kappa B and AP-2 consensus binding sites. The presence of regulatory sequences for the binding of transcription factors such as NF-kappa B and AP-2, whose activation is associated with the immediate response of the cell to an injury, may be an indication of the important role which HO-1 may play in defense mechanisms against tissue injury.

Heme is essential for cell respiration, energy generation, and oxidative biotransformations. The latter function is exemplified by the oxidative metabolism of various endogenous and exogenous chemicals catalyzed by the heme protein cytochrome P-450. Recent studies have established that metal ions directly regulate cellular content of heme, and thus of heme proteins by controlling production of delta-aminolevulinate synthetase and heme oxygenase, the rate-limiting enzymes for heme synthesis and degradation, respectively. Metal ions also alter cellular content of glutathione. In excess amounts, metal ions greatly accelerate the turnover and degradation of heme and substantially impair the oxidative functions of cells--particularly those dependent on cytochrone P-450. As a result, the biological impact of chemicals which are detoxified or metabolically transformed by the P-450 system is greatly altered.

Aspirin and other NSAIDs reduce the incidence of and mortality from colon cancer, but their mechanism of action remains unknown. We evaluated the effect of aspirin (ASA) and three other structurally unrelated NSAIDs (indomethacin, naproxen, and piroxicam) on cell proliferation, cell cycle phase distribution, and the development of apoptosis in HT-29 colon adenocarcinoma cells in vitro. All of the NSAIDs examined reduced the proliferation and altered the morphology of these cells in a time- and concentration-dependent manner. In addition, they altered the cell cycle phase distribution of these cells. They increased the proportion of cells in the G0/G1 phase and reduced the proportion in the S phase of the cell cycle. ASA and indomethacin also reduced the percentage of cells in the G2/M phase, whereas naproxen and piroxicam did not. Parallel to their effect on cell cycle, ASA and indomethacin also reduced the levels of p34cdc2 and p33cdk2, two cyclin-dependent kinases that are important for cell cycle progression. Finally, all the NSAIDs analyzed, except ASA, induced apoptosis in these cells. There as a rough correlation between the relative potency of these compounds in inducing apoptosis and their effectiveness in retarding cell proliferation. Our findings indicate that NSAIDs can reduce the proliferation of HT-29 colon cancer cells in vitro. In addition, they cause cell cycle quiescence and apoptosis, both of which could account for their anti-proliferative effect. These findings suggest possible mechanisms for the cancer preventive effects of these compounds in humans.

Studies were carried out to examine the effects of dietary fat and cholesterol on cholesterol homeostasis in man. 75 12-wk studies were carried out during intake of 35% of calories as either saturated or polyunsaturated fat, first low and then high in dietary cholesterol. Dietary fat and cholesterol intakes, plasma lipid and lipoprotein levels, cholesterol absorption and sterol synthesis in isolated blood mononuclear leukocytes were measured during each diet period. In 69% of the studies the subjects compensated for the increased cholesterol intake by decreasing cholesterol fractional absorption and/or endogenous cholesterol synthesis. When an increase in plasma cholesterol levels was observed there was a failure to suppress endogenous cholesterol synthesis. Plasma cholesterol levels were more sensitive to dietary fat quality than to cholesterol quantity. The results demonstrate that the responses to dietary cholesterol and fat are highly individualized and that most individuals have effective feedback control mechanisms.

Cc blood function 80 per min.

The effects of various metalloporphyrins on hepatic heme oxygenase (EC 1.14.99.3) activity were examined in order to identify compounds that could inhibit heme degradation to bile pigment and might therefore be utilized to suppress the development of hyperbilirubinemia in the newborn. Among nine metal-protoporphyrin IX chelates (i.e., metal-hemes) studied, Sn-heme, Mn-heme, and Zn-heme substantially diminished heme oxygenase activity in vivo in the rat. These metalloporphyrins act as competitive inhibitory substrates in the heme oxygenase reaction but are not themselves oxidatively degraded. Sn-heme was the most potent enzyme inhibitor (Ki = 0.011 microM) in liver, spleen, kidney, and skin. Sn-heme administered to newborn animals within the first 72 hr after birth blocked the postnatal increase in heme oxygenase activity that occurs in various tissues. Its effect on the enzyme levels was prompt and protracted. Sn-heme administration also entirely prevented the development of hyperbilirubinemia that normally occurs postnatally. The effect of the metalloporphyrin in lowering the increased concentrations of serum bilirubin in neonates was prompt (within 1 day) and persisted throughout the 42 days after birth. No deleterious effects of Sn-heme treatment of the newborn were observed. This demonstrates that a synthetic metalloporphyrin that is a potent competitive inhibitor of heme oxidation can, when administered to the newborn, also prevent the hyperbilirubinemia that normally develops postnatally. The potential clinical implications of these findings are evident, and it is suggested that the pharmacological properties of Sn-heme and related synthetic metalloporphyrins merit further study.

Summary A technique has been developed which allows reliable quantitative assessment of phagocytosis of bacteria by rabbit granulocytes in vitro, and which permits study of the role of heat-labile serum factors in the phagocytic process. Incubation of bacteria with fresh serum opsonized the organisms. After washing they were fully susceptible to ingestion in a serum-free system. Using this preincubation technique the influence of ionic composition of the medium was observed on the opsonization and on the ingestion processes separately. High concentration of salt blocked both processes. Divalent cations were required for ingestion but not for opsonization of the bacteria. The lack of requirement for calcium and magnesium speaks against identification of heat-labile serum opsonin with the complete hemolytic complement system. Features of heat-labile serum opsonin which distinguished it from antibody were the following: a) heat-lability, b) titer for various bacteria remarkably constant in various serum specimens, c) rapid reaction with bacteria at 38°C but not at 0°C, d) destruction by hydrazine or ammonia, e) not γ globulin, and f) lack of specificity as demonstrated in adsorption studies.

Mature human dendritic cells can be generated in substantial numbers from nonproliferating progenitors in human blood using a two-step protocol. T cell-depleted mononuclear cells are first cultured with granulocyte-macrophage colony-stimulating factor and interleukin-4 (IL-4) and then exposed to monocyte conditioned medium (MCM). The dendritic cells generated using this approach are rendered terminally mature and are the most potent antigen presenting cells identified to date in humans. We sought to characterize factors in MCM that induce the terminal differentiation of dendritic cells. MCM contained substantial, although varying, quantities of several factors including tumor necrosis factor-alpha, IL-1beta, IL-6, and interferon-alpha. However, none of the four factors, individually or in various combinations, could fully substitute for the MCM to generate irreversibly differentiated dendritic cells. The yields, percentage of cells expressing the mature phase marker CD83, and mixed leukocyte reaction-stimulatory function were lower when defined cytokines were used in the place of MCM. Therefore, the full maturation of dendritic cells, because it entails changes in many known cell and molecular properties, requires a number of different cytokines that are released in tandem from appropriately stimulated monocytes. We propose that MCM-matured dendritic cells will be the most effective adjuvants for immunotherapy in vivo.

Ten healthy volunteers were fed (1) a control diet for 10 days, (2) the same control diet for 3 additional days and a brussels sprouts and cabbage‐containing diet for the next 7 days, and (3) the control diet for the 10 subsequent days. Antipyrine and phenacetin were administered orally before breakfast on days 7 and 10, respectively, of each dietary regimen. During the test diet period the mean plasma half‐life of antipyrine decreased 13% and the mean metabolic clearance rate increased 11%. While small, these changes were statistically significant, indicating a stimulatory effect of cabbage and brussels sprouts on antipyrine metabolism. The values returned to control levels during the second control diet period. During the test diet period the mean plasma concentration of phenacetin was decreased by 34% to 67% at each time interval from 0.5 to 7 hr after phenacetin administration, and the mean plasma concentration of total (conjugated plus unconjugated) N‐acetyl‐p‐aminophenol (APAP), phenacetin's major metabolite, was increased from 0.5 to 3 hr after phenacetin administration. The concentrations of phenacetin and APAP returned toward control values during the second control diet period. The mean plasma half‐life of phenacetin was not influenced by the dietary changes. These results suggest that the test diet enhanced the metabolism of phenacetin in the gastrointestinal tract and/or during its first pass through the liver. Feeding the test diet increased the mean ratio of conjugated APAP to unconjugated APAP observed in plasma at each time interval from 0.5 to 7 hr after phenacetin administration, suggesting that the diet enhanced the conjugation of APAP.

Optimal HIV vaccines should elicit CD8+ T cells specific for HIV proteins presented on MHC class I products, because these T cells contribute to host resistance to viruses. We had previously found that the targeting of antigen to dendritic cells (DCs) in mice efficiently induces CD8+ T cell responses. To extend this finding to humans, we introduced the HIV p24 gag protein into a mAb that targets DEC-205/CD205, an endocytic receptor of DCs. We then assessed cross-presentation, which is the processing of nonreplicating internalized antigen onto MHC class I for recognition by CD8+ T cells. Low doses of alphaDEC-gag, but not control Ig-gag, stimulated proliferation and IFN-gamma production by CD8+ T cells isolated from the blood of HIV-infected donors. alphaCD205 fusion mAb was more effective for cross-presentation than alphaCD209/DC-SIGN, another abundant DC uptake receptor. Presentation was diverse, because we identified eight different gag peptides that were recognized via DEC-205 in 11 individuals studied consecutively. Our results, based on humans with highly polymorphic MHC products, reveal that DCs and DEC-205 can cross-present several different peptides from a single protein. Because of the consistency in eliciting CD8+ T cell responses, these data support the testing of alphaDEC-205 fusion mAb as a protein-based vaccine.

SummarySix subjects with obesity of exogenous origin showed significant reductions in serum concentrations of free and esterified cholesterol and of phospholipids when plant fats were substituted isocalorically for animal fats during a 4-month metabolic ward study. The approximate magnitude of the change was 20% decrease. Neutral fat levels showed no significant change. Body weights and caloric intakes were held constant throughout the experiment.

Clinical success in rehabilitation of heroin addicts with maintenance treatment requires stability of the blood level in a pharmacologically effective range (optimally, 150 to 600 ng/mL)-a phenomenon that emphasizes the central importance of narcotic receptor occupation. It is postulated that the high rate of relapse of addicts after detoxification from heroin use is due to persistent derangement of the endogenous ligand-narcotic receptor system and that methadone in an adequate daily dose compensates for this defect. Some patients with long histories of heroin use and subsequent rehabilitation on a maintenance program do well when the treatment is terminated. The majority, unfortunately, experience a return of symptoms after maintenance is stopped. The treatment, therefore, is corrective but not curative for severely addicted persons. A major challenge for future research is to identify the specific defect in receptor function and to repair it. Meanwhile, methadone maintenance provides a safe and effective way to normalize the function of otherwise intractable narcotic addicts.