We have employed a method based on the Cre-loxP recombination system of bacteriophage P1 to generate a mouse strain in which the JH segments and the intron enhancer in the IgH locus are deleted. By analysis of immunoglobulin isotype switch recombination in heterozygous mutant B cells activated by lipopolysaccharide plus interleukin-4, we show that, on the mutant chromosome, switch recombination at the mu gene switch region is strongly suppressed, whereas the switch region of the gamma 1 gene is efficiently rearranged. These data demonstrate an independent control of switch recombination at individual switch regions and suggest that, in the process of switch recombination, the alignment of the recombining strands occurs independently of and probably after the introduction of double-strand breaks into the switch regions involved.

Rheumatoid arthritis (RA) is a widely prevalent (1-3%) chronic systemic disease thought to have an autoimmune component; both humoral and cellular mechanisms have been implicated. Primary osteoarthritis (OA) is considered to be distinct from rheumatoid arthritis, and here damage is thought to be secondary to cartilage degeneration. In rheumatoid arthritis, immune complexes are present that consist exclusively of immunoglobulin, implying that this is both the 'antibody' (rheumatoid factor [RF]) and the 'antigen' (most commonly IgG). Autoantigenic reactivity has been localized to the constant-region (C gamma 2) domains of IgG. There is no evidence for a polypeptide determinant but carbohydrate changes have been reported. We have therefore conducted a study, simultaneously in Oxford and Tokyo, to compare in detail the N-glycosylation pattern of serum IgG (Fig. 1) isolated from normal individuals and from patients with either primary osteoarthritis or rheumatoid arthritis. The results, which required an evaluation of the primary sequences of approximately 1,400 oligosaccharides from 46 IgG samples, indicate that: (1) IgG isolated from normal individuals, patients with RA and patients with OA contains different distributions of asparagine-linked bi-antennary complex-type oligosaccharide structures, (2) in neither disease is the IgG associated with novel oligosaccharide structures, but the observed differences are due to changes in the relative extent of galactosylation compared with normal individuals. This change results in a 'shift' in the population of IgG molecules towards those carrying complex oligosaccharides, one or both of whose arms terminate in N-acetylglucosamine. These two arthritides may therefore be glycosylation diseases, reflecting changes in the intracellular processing, or post-secretory degradation of N-linked oligosaccharides.

At least two types of somatic recombination are necessary for the generation of a complete immunoglobulin gamma 2b gene from germ-line DNA sequences. The first type of recombination consists of the assembly of three separate DNA segments, each encoding a different part of the variable region. The second type of recombination replaces the exons coding for the constant region of the mu chain with those coding for the same region of the gamma 2b chain. The DNA sequencing studies suggest that the two types of recombination operate by different mechanisms.

A window of opportunity for immune responses to extinguish human immunodeficiency virus type 1 (HIV-1) exists from the moment of transmission through establishment of the latent pool of HIV-1-infected cells. A critical time to study the initial immune responses to the transmitted/founder virus is the eclipse phase of HIV-1 infection (time from transmission to the first appearance of plasma virus), but, to date, this period has been logistically difficult to analyze. To probe B-cell responses immediately following HIV-1 transmission, we have determined envelope-specific antibody responses to autologous and consensus Envs in plasma donors from the United States for whom frequent plasma samples were available at time points immediately before, during, and after HIV-1 plasma viral load (VL) ramp-up in acute infection, and we have modeled the antibody effect on the kinetics of plasma viremia. The first detectable B-cell response was in the form of immune complexes 8 days after plasma virus detection, whereas the first free plasma anti-HIV-1 antibody was to gp41 and appeared 13 days after the appearance of plasma virus. In contrast, envelope gp120-specific antibodies were delayed an additional 14 days. Mathematical modeling of the earliest viral dynamics was performed to determine the impact of antibody on HIV replication in vivo as assessed by plasma VL. Including the initial anti-gp41 immunoglobulin G (IgG), IgM, or both responses in the model did not significantly impact the early dynamics of plasma VL. These results demonstrate that the first IgM and IgG antibodies induced by transmitted HIV-1 are capable of binding virions but have little impact on acute-phase viremia at the timing and magnitude that they occur in natural infection.

Almost all of the key molecules involved in the innate and adaptive immune response are glycoproteins. In the cellular immune system, specific glycoforms are involved in the folding, quality control, and assembly of peptide-loaded major histocompatibility complex (MHC) antigens and the T cell receptor complex. Although some glycopeptide antigens are presented by the MHC, the generation of peptide antigens from glycoproteins may require enzymatic removal of sugars before the protein can be cleaved. Oligosaccharides attached to glycoproteins in the junction between T cells and antigen-presenting cells help to orient binding faces, provide protease protection, and restrict nonspecific lateral protein-protein interactions. In the humoral immune system, all of the immunoglobulins and most of the complement components are glycosylated. Although a major function for sugars is to contribute to the stability of the proteins to which they are attached, specific glycoforms are involved in recognition events. For example, in rheumatoid arthritis, an autoimmune disease, agalactosylated glycoforms of aggregated immunoglobulin G may induce association with the mannose-binding lectin and contribute to the pathology.

Two major components are required for a successful prediction of the three-dimensional structure of peptides and proteins: an efficient global optimization procedure which is capable of finding an appropriate local minimum for the strongly anisotropic function of hundreds of variables, and a set of free energy components for a protein molecule in solution which are computationally inexpensive enough to be used in the search procedure, yet sufficiently accurate to ensure the uniqueness of the native conformation. We here found an efficient way to make a random step in a Monte Carlo procedure given knowledge of the energy or statistical properties of conformational subspaces (e.g. phi-psi zones or side-chain torsion angles). This biased probability Monte Carlo (BPMC) procedure randomly selects the subspace first, then makes a step to a new random position independent of the previous position, but according to the predefined continuous probability distribution. The random step is followed by a local minimization in torsion angle space. The positions, sizes and preferences for high-probability zones on phi-psi maps and chi-angle maps were calculated for different residue types from the representative set of 191 and 161 protein 3D-structures, respectively. A fast and precise method to evaluate the electrostatic energy of a protein in solution is developed and combined with the BPMC procedure. The method is based on the modified spherical image charge approximation, efficiently projected onto a molecule of arbitrary shape. Comparison with the finite-difference solutions of the Poisson-Boltzmann equation shows high accuracy for our approach. The BPMC procedure is applied successfully to the structure prediction of 12- and 16-residue synthetic peptides and the determination of protein structure from NMR data, with the immunoglobulin binding domain of streptococcal protein G as an example. The BPMC runs display much better convergence properties than the non-biased simulations. The advantage of a true global optimization procedure for NMR structure determination is its ability to cope with local minima originating from data errors and ambiguities in NMR data.

BACKGROUND

The prevalence of monoclonal gammopathy of undetermined significance (MGUS), a premalignant plasma-cell disorder, among persons 50 years of age or older has not been accurately determined. We used sensitive laboratory techniques to ascertain the prevalence of MGUS in a large population in a well-defined geographic area.

METHODS

We identified all living residents of Olmsted County, Minnesota, as of January 1, 1995. We obtained serum that remained after the performance of routine clinical tests at Mayo Clinic or asked subjects for whom such serum was unavailable to provide a sample. Agarose-gel electrophoresis was performed on all serum samples, and any serum sample with a discrete band of monoclonal protein or thought to have a localized band was subjected to immunofixation.

RESULTS

Serum samples were obtained from 21,463 of the 28,038 enumerated residents 50 years of age or older (76.6 percent). MGUS was identified in 694 (3.2 percent) of these persons. Age-adjusted rates were higher in men than in women (4.0 percent vs. 2.7 percent, P<0.001). The prevalence of MGUS was 5.3 percent among persons 70 years of age or older and 7.5 percent among those 85 years of age or older. The concentration of monoclonal immunoglobulin was less than 1.0 g per deciliter in 63.5 percent and at least 2.0 g per deciliter in only 4.5 percent of 694 persons. The concentration of uninvolved immunoglobulins was reduced in 27.7 percent of 447 persons tested, and 21.5 percent of 79 tested had a monoclonal urinary light chain.

CONCLUSIONS

Among residents of Olmsted County, Minnesota, MGUS was found in 3.2 percent of persons 50 years of age or older and 5.3 percent of persons 70 years of age or older.

A sensitive assay was used to measure the binding of iodine-125-labeled insulin in serum obtained from 112 newly diagnosed insulin-dependent diabetics before insulin treatment was initiated. Two groups of nondiabetics served as controls: children with a variety of diseases other than diabetes and nondiabetic siblings of insulin-dependent diabetics. Eighteen of the diabetics were found to have elevated binding and 36 were above the 95th percentile of control values. The insulin-binding protein is precipitated by antibody to human immunoglobulin G, has a displacement curve that is parallel and over the same concentration range as serum from long-standing insulin-dependent diabetics, and elutes from a Sephacryl S-300 column at the position of gamma globulin. These insulin antibodies are present in a large percentage of newly diagnosed, untreated diabetics and may be an immune marker of B-cell damage.

BACKGROUND

Cytomegalovirus (CMV) is a leading cause of congenital illness and disability, including hearing loss and mental retardation. However, there are no nationwide estimates of CMV seroprevalence among pregnant women or the overall population of the United States.

METHODS

To determine CMV prevalence in a representative sample of the US population, we tested serum samples for CMV-specific immunoglobulin G from participants aged>> or =6 years (n=21,639) in the third National Health and Nutrition Examination Survey (1988-1994).

RESULTS

The prevalence of CMV infection was 58.9% in individuals>> or =6 years old. CMV seroprevalence increased gradually with age, from 36.3% in 6-11-year-olds to 90.8% in those aged>> or =80 years. CMV seroprevalence differed by race and/or ethnicity as follows: 51.2% in non-Hispanic white persons, 75.8% in non-Hispanic black persons, and 81.7% in Mexican Americans. Racial and/or ethnic differences in CMV seroprevalence persisted when controlling for household income level, education, marital status, area of residence, census region, family size, country of birth, and type of medical insurance. Among women, racial and/or ethnic differences were especially significant; between ages 10-14 years and 20-24 years, seroprevalence increased 38% for non-Hispanic black persons, 7% for non-Hispanic white persons, and <1% for Mexican Americans.

CONCLUSIONS

On the basis of these results, we estimate that each year in the United States approximately 340,000 non-Hispanic white persons, 130,000 non-Hispanic black persons, and 50,000 Mexican American women of childbearing age experience a primary CMV infection. Given the number of women at risk and the significance of congenital disease, development of programs for the prevention of CMV infection, such as vaccination or education, is of considerable public health importance.

The mechanism responsible for immunoglobulin class switch recombination is unknown. Previous work has shown that class switch sequences have the unusual property of forming RNA-DNA hybrids when transcribed in vitro. Here we show that the RNA-DNA hybrid structure that forms in vitro is an R-loop with a displaced guanine (G)-rich strand that is single-stranded. This R-loop structure exists in vivo in B cells that have been stimulated to transcribe the gamma3 or the gamma2b switch region. The length of the R-loops can exceed 1 kilobase. We propose that this distinctive DNA structure is important in the class switch recombination mechanism

The regulation of the subclass of immunoglobulin secreted by B cells has been studied in vitro in polyclonal systems using mitogens, such as lipopolysaccharide (LPS), to bypass the requirement for cognate interaction between antigen-specific T and B cells. In these systems, interleukin-(IL)-4 induces the secretion of IgG1 (ref. 1) and IgE (ref. 2); IL-5 enhances the secretion of IgA, and interferon-gamma (IFN-gamma) enhances the secretion of IgG2a (ref. 5). Clones of murine TH cells can be divided into two subsets, TH1 and TH2 (ref. 6). Both subsets synthesize IL-3 and granulocyte-monocyte colony-stimulating factor (GM-CSF), but only TH1 clones produce IL-2, IFN-gamma, and lymphotoxin (LT) and TH2 clones produce IL-4 and IL-5 (ref. 7). We have examined the role of clones of antigen-specific TH1 and TH2 cells in the regulation of the subclasses of IgG antibody secreted by antigen-specific B cells. Our results show that both types of TH cells induce the secretion of IgM and IgG3, whereas clones of TH1 and TH2 cells specifically induce antigen-specific B cells to secrete IgG2a and IgG1, respectively. We also demonstrate that regulation of commitment to the secretion of a particular IgG isotype occurs in two distinct stages: cognate interaction between T and B cells and interaction between T-cell-derived lymphokines and B cells.

Recent investigations have suggested that tissue-specific regulatory factors are required for immunoglobulin gene transcription. Cells of the mouse lymphocytoid pre-B-cell line 70Z/3 contain a constitutively rearranged immunoglobulin kappa light chain gene; the nucleotide sequence of this gene exhibits all the known properties of a functionally competent transcription unit. Nevertheless, transcripts derived from this gene are detectable only after exposure of the cells to bacterial lipopolysaccharide, implying that accurate DNA rearrangement is not sufficient to activate expression of the gene. Comparison of the sequence of the 70Z/3 kappa light chain gene with those encoding other immunoglobulin heavy and light chains has revealed that a distinctive promoter region structure is characteristic of this multigene family. The sequence A-T-T-T-G-C-A-T lies approximately 70 base pairs upstream from the site of transcriptional initiation in every light chain gene examined; in heavy chain genes, the corresponding location is occupied by the precise inverse (A-T-G-C-A-A-A-T) of this sequence. Although adjacent regions of DNA have diverged extensively in evolution, these octanucleotide sequences are stringently conserved at this location among diverse immunoglobulin genes from at least two mammalian species. The proximity of this conserved octanucleotide block to the site of transcriptional initiation suggests that it may serve as a recognition locus for factors regulating immunoglobulin gene expression in a tissue-specific fashion.

Dendritic cells (DCs) express several receptors for the Fc portion of immunoglobulin (Ig)G (FcgammaR), which mediate internalization of antigen-IgG complexes (immune complexes, ICs) and promote efficient major histocompatibility complex (MHC) class II-restricted antigen presentation. We now show that FcgammaRs have two additional specific attributes in murine DCs: the induction of DC maturation and the promotion of efficient MHC class I-restricted presentation of peptides from exogenous, IgG-complexed antigens. Both FcgammaR functions require the FcgammaR-associated gamma chain. FcgammaR-mediated MHC class I-restricted antigen presentation is extremely sensitive and specific to immature DCs. It requires proteasomal degradation and is dependent on functional peptide transporter associated with antigen processing, TAP1-TAP2. By promoting DC maturation and presentation on both MHC class I and II molecules, ICs should efficiently sensitize DCs for priming of both CD4(+) helper and CD8(+) cytotoxic T lymphocytes in vivo.

It is well established that high doses of monomeric immunoglobulin G (IgG) purified from pooled human plasma [intravenous immunoglobulin (IVIG)] confer anti-inflammatory activity in a variety of autoimmune settings. However, exactly how those effects are mediated is not clear because of the heterogeneity of IVIG. Recent studies have demonstrated that the anti-inflammatory activity of IgG is completely dependent on sialylation of the N-linked glycan of the IgG Fc fragment. Here we determine the precise glycan requirements for this anti-inflammatory activity, allowing us to engineer an appropriate IgGGG and helps guide development of an IVIG replacement with improved activity and availability.

In Plasmodium falciparum-endemic areas, pregnancy-associated malaria (PAM) is an important health problem. The condition is precipitated by accumulation of parasite-infected erythrocytes (IEs) in the placenta, and this process is mediated by parasite-encoded variant surface antigens (VSA) binding to chondroitin sulfate A (CSA). Parasites causing PAM express unique VSA types, VSAPAM, which can be serologically classified as sex specific and parity dependent. It is sex specific because men from malaria-endemic areas do not develop VSAPAM antibodies; it is parity dependent because women acquire anti-VSAPAM immunoglobulin (Ig) G as a function of parity. Previously, it was shown that transcription of var2csa is up-regulated in placental parasites and parasites selected for CSA binding. Here, we show the following: (a) that VAR2CSA is expressed on the surface of CSA-selected IEs; (b) that VAR2CSA is recognized by endemic plasma in a sex-specific and parity-dependent manner; (c) that high anti-VAR2CSA IgG levels can be found in pregnant women from both West and East Africa; and (d) that women with high plasma levels of anti-VAR2CSA IgG give birth to markedly heavier babies and have a much lower risk of delivering low birth weight children than women with low levels.

BACKGROUND

X-linked severe combined immunodeficiency due to a mutation in the gene encoding the common gamma (gamma(c)) chain is a lethal condition that can be cured by allogeneic stem-cell transplantation. We investigated whether infusion of autologous hematopoietic stem cells that had been transduced in vitro with the gamma(c) gene can restore the immune system in patients with severe combined immunodeficiency.

METHODS

CD34+ bone marrow cells from five boys with X-linked severe combined immunodeficiency were transduced ex vivo with the use of a defective retroviral vector. Integration and expression of the gamma(c) transgene and development of lymphocyte subgroups and their functions were sequentially analyzed over a period of up to 2.5 years after gene transfer.

RESULTS

No adverse effects resulted from the procedure. Transduced T cells and natural killer cells appeared in the blood of four of the five patients within four months. The numbers and phenotypes of T cells, the repertoire of T-cell receptors, and the in vitro proliferative responses of T cells to several antigens after immunization were nearly normal up to two years after treatment. Thymopoiesis was documented by the presence of naive T cells and T-cell antigen-receptor episomes and the development of a normal-sized thymus gland. The frequency of transduced B cells was low, but serum immunoglobulin levels and antibody production after immunization were sufficient to avoid the need for intravenous immunoglobulin. Correction of the immunodeficiency eradicated established infections and allowed patients to have a normal life.

CONCLUSIONS

Ex vivo gene therapy with gamma(c) can safely correct the immune deficiency of patients with X-linked severe combined immunodeficiency.

Aberrant glycosylation expressed in glycosphingolipids and glycoproteins in tumor cells has been implicated as an essential mechanism in defining stage, direction, and fate of tumor progression. This general concept is supported by results from three lines of study: (a) Numerous clinicopathological studies have shown a clear correlation between aberrant glycosylation status of primary tumor and invasive/metastatic potential of human cancer as reflected by 5- or 10-year survival rates of patients. (b) Carbohydrates expressed in tumor cells are either adhesion molecules per se or modulate adhesion receptor function. Some are directly involved in cell adhesion. They are recognized by selectins or other carbohydrate-binding proteins or by complementary carbohydrates (through carbohydrate-carbohydrate interaction). N- or O-glycosylation of functionally important membrane components may alter tumor cell adhesion or motility in a direction that either promotes or inhibits invasion and metastasis. Examples of such receptors are E-cadherin, integrins, immunoglobulin family receptors (e.g., CD44), and lysosome-associated membrane protein. (c) Gangliosides and sphingolipids modulate transmembrane signaling essential for tumor cell growth, invasion, and metastasis. The transducer molecules susceptible to gangliosides and sphingolipids include integrin receptors, tyrosine kinase-linked growth factor receptors, protein kinase C, and G-protein-linked receptor affecting protein kinase A. Some glycosphingolipids (e.g., Gb3Cer, Le(y), ceramide, and sphingosine induce tumor cell differentiation and subsequent apoptosis. Shedded gangliosides may block immunogenicity of tumor cells, providing conditions favorable for "escape" from immunological suppression of tumor growth by the host. Various reagents that block carbohydrate-mediated tumor cell adhesion or block glycosylation processing have been shown to inhibit tumor cell metastasis. This provides the basis for further development of "anti-adhesion therapy." Ganglioside analogues and sphingolipid analogues that inhibit protein kinase C and receptor-associated tyrosine kinase have been applied for inhibition of metastasis. A crucial mechanism for inhibition of metastasis by these reagents may involve blocking of transmembrane signaling for expression of P- and E-selectin. This provides the basis for development of "ortho-signaling therapy."

To date, more than 20 recombinant immunoglobulin G (IgG) antibody therapeutics are licensed for the treatment of various diseases. The mechanism of action of recombinant monoclonal antibodies (rMAbs) has been extensively investigated and several distinct pathways have been defined; selective activation of specific pathways may optimize clinical outcomes for different diseases, such as cancer and chronic inflammation. Human IgG is a glycoprotein with oligosaccharides attached at a single site. These are essential to the mode of action of rMAbs, and the antibody efficacy can vary depending on the particular oligosaccharide that is attached. Methods are now becoming available that allow the production of rMAbs bearing pre-selected oligosaccharides - glycoforms - to provide maximum efficacy for a given disease indication. This Review summarizes current knowledge of these methods and avenues for their exploitation in the clinic.

Despite its widespread distribution on both lymphoid and myeloid cells, the biological role of the low-affinity immunoglobulin-G receptor, Fc gamma RII, is not fully understood. Defects in this receptor or its signalling pathway in B cells result in perturbations in immune-complex-mediated feedback inhibition of antibody production. We now report that Fc gamma RII-deficient animals display elevated immunoglobulin levels in response to both thymus-dependent and thymus-independent antigens. Additionally, the effector arm of the allergic response is perturbed in these mice. Mast cells from Fc gamma RII-/- are highly sensitive to IgG-triggered degranulation, in contrast to their wild-type counterparts. Fc gamma RII-deficient mice demonstrate an enhanced passive cutaneous analphylaxis reaction, the result of a decreased threshold for mast-cell activation by Fc gamma RIII cross-linking. These results demonstrate that Fc gamma RII acts as a general negative regulator of immune-complex-triggered activation in vivo for both the afferent and efferent limbs of the immune response. Exploiting this property offers new therapeutic opportunities for the treatment of allergic and autoimmune disorders.

The presence of hepatitis C virus (HCV) RNA-containing particles in the low-density fractions of plasma has been associated with high infectivity. However, the nature of circulating HCV particles and their association with immunoglobulins or lipoproteins as well as the characterization of cell entry have all been subject to conflicting reports. For a better analysis of HCV RNA-containing particles, we quantified HCV RNA in the low-density fractions of plasma corresponding to the very-low-density lipoprotein (VLDL), intermediate-density lipoprotein, and low-density lipoprotein (LDL) fractions from untreated chronically HCV-infected patients. HCV RNA was always found in at least one of these fractions and represented 8 to 95% of the total plasma HCV RNA. Surprisingly, immunoglobulins G and M were also found in the low-density fractions and could be used to purify the HCV RNA-containing particles (lipo-viro-particles [LVP]). Purified LVP were rich in triglycerides; contained at least apolipoprotein B, HCV RNA, and core protein; and appeared as large spherical particles with a diameter of more than 100 nm and with internal structures. Delipidation of these particles resulted in capsid-like structures recognized by anti-HCV core protein antibody. Purified LVP efficiently bind and enter hepatocyte cell lines, while serum or whole-density fractions do not. Binding of these particles was competed out by VLDL and LDL from noninfected donors and was blocked by anti-apolipoprotein B and E antibodies, whereas upregulation of the LDL receptor increased their internalization. These results suggest that the infectivity of LVP is mediated by endogenous proteins rather than by viral components providing a mechanism of escape from the humoral immune response.

The in vitro analysis of intestinal epithelium has been hampered by a lack of suitable culture systems. Here we describe robust long-term methodology for small and large intestinal culture, incorporating an air-liquid interface and underlying stromal elements. These cultures showed prolonged intestinal epithelial expansion as sphere-like organoids with proliferation and multilineage differentiation. The Wnt growth factor family positively regulates proliferation of the intestinal epithelium in vivo. Accordingly, culture growth was inhibited by the Wnt antagonist Dickkopf-1 (Dkk1) and markedly stimulated by a fusion protein between the Wnt agonist R-spondin-1 and immunoglobulin Fc (RSpo1-Fc). Furthermore, treatment with the gamma-secretase inhibitor dibenzazepine and neurogenin-3 overexpression induced goblet cell and enteroendocrine cell differentiation, respectively, consistent with endogenous Notch signaling and lineage plasticity. Epithelial cells derived from both leucine-rich repeat-containing G protein-coupled receptor-5-positive (Lgr5(+)) and B lymphoma moloney murine leukemia virus insertion region homolog-1-positive (Bmi1(+)) lineages, representing putative intestinal stem cell (ISC) populations, were present in vitro and were expanded by treatment with RSpo1-Fc; this increased number of Lgr5(+) cells upon RSpo1-Fc treatment was subsequently confirmed in vivo. Our results indicate successful long-term intestinal culture within a microenvironment accurately recapitulating the Wnt- and Notch-dependent ISC niche.

BACKGROUND

Alzheimer's disease is characterized by the presence of cortical amyloid-beta (Aβ) protein plaques, which result from the sequential action of β-secretase and γ-secretase on amyloid precursor protein. Semagacestat is a small-molecule γ-secretase inhibitor that was developed as a potential treatment for Alzheimer's disease.

METHODS

We conducted a double-blind, placebo-controlled trial in which 1537 patients with probable Alzheimer's disease underwent randomization to receive 100 mg of semagacestat, 140 mg of semagacestat, or placebo daily. Changes in cognition from baseline to week 76 were assessed with the use of the cognitive subscale of the Alzheimer's Disease Assessment Scale for cognition (ADAS-cog), on which scores range from 0 to 70 and higher scores indicate greater cognitive impairment, and changes in functioning were assessed with the Alzheimer's Disease Cooperative Study-Activities of Daily Living (ADCS-ADL) scale, on which scores range from 0 to 78 and higher scores indicate better functioning. A mixed-model repeated-measures analysis was used.

RESULTS

The trial was terminated before completion on the basis of a recommendation by the data and safety monitoring board. At termination, there were 189 patients in the group receiving placebo, 153 patients in the group receiving 100 mg of semagacestat, and 121 patients in the group receiving 140 mg of semagacestat. The ADAS-cog scores worsened in all three groups (mean change, 6.4 points in the placebo group, 7.5 points in the group receiving 100 mg of the study drug, and 7.8 points in the group receiving 140 mg; P=0.15 and P=0.07, respectively, for the comparison with placebo). The ADCS-ADL scores also worsened in all groups (mean change at week 76, -9.0 points in the placebo group, -10.5 points in the 100-mg group, and -12.6 points in the 140-mg group; P=0.14 and P<0.001, respectively, for the comparison with placebo). Patients treated with semagacestat lost more weight and had more skin cancers and infections, treatment discontinuations due to adverse events, and serious adverse events (P<0.001 for all comparisons with placebo). Laboratory abnormalities included reduced levels of lymphocytes, T cells, immunoglobulins, albumin, total protein, and uric acid and elevated levels of eosinophils, monocytes, and cholesterol; the urine pH was also elevated.

CONCLUSIONS

As compared with placebo, semagacestat did not improve cognitive status, and patients receiving the higher dose had significant worsening of functional ability. Semagacestat was associated with more adverse events, including skin cancers and infections. (Funded by Eli Lilly; ClinicalTrials.gov number, NCT00594568.)

Suspension cultures of a human monocytic leukemia cell line, THP-1, were treated with 0.16 to 160 nM 12-O-tetradecanoylphorbol-13-acetate (TPA). In an original cell line, THP-1-O, cultured again from -80 degrees cryopreservation, more than 80% of the cells adhered to the glass substrate with marked morphological change within 3 hr of TPA treatment. Adherent cells became flat and amoeboid in shape, and many microvilli and flaps of the cell surface disappeared. Well-developed Golgi apparatus, rough endoplasmic reticula, and a large amount of free ribosomes were seen in the cytoplasm. On the other hand, in THP-1-R cells cultured continuously without cryopreservation for 26 months, approximately 80% of the cells adhered to the substrate 48 hr after TPA treatment. Round and ovoid shapes were kept in THP-1-R cells treated with TPA. Surface Fc receptors for immunoglobulin G were present on more than 90% of THP-1-O and THP-1-R cells and were little affected by treatment with TPA. Sixty to 70% of the TPA-treated THP-1-O and THP-1-R cells were able to phagocytize yeasts and immunoglobulin G-coated sheep erythrocytes. Less than 20% of the untreated THP-1 cells were able to phagocytize yeasts and immunoglobulin G-coated sheep erythrocytes. In histochemical staining, alpha-naphthyl butyrate esterase was enhanced after treatment with TPA. Lysozyme activity in culture supernatants was not affected by TPA treatment. When exposed to latex beads and TPA, increased 14CO2 production from [1-14C]glucose in THP-1-O cells was observed. These results indicate that, after treatment with TPA, human monocytic leukemia cells may be converted into mature cells with functions of macrophages.