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Papers by Daudi Langat

Gynecologic and Obstetric Investigation, Feb 1, 2004

Journal of Ethnopharmacology, Feb 20, 2006

The potential effect of Khat (Catha edulis, Celastraceae) on fertility in humans has not been elu... more The potential effect of Khat (Catha edulis, Celastraceae) on fertility in humans has not been elucidated. In this study, we used the olive baboon (Papio anubis, Cercopithecidae) to determine the effects of oral administration of khat on circulating hormones. In order to establish baseline hormonal levels, five male baboons were bled once a week for 1 month. The same baboons were then fed with crude khat juice extract once a week over a period of 2 months, and the effects on serum levels of cortisol, testosterone and prolactin determined using enzyme immunoassay (EIA) and radioimmunoassay (RIA). Subsequently, sampling was repeated for a further 1 month to determine the residual effect of khat. The results showed that khat administration causes a significant increase in the mean levels of testosterone while prolactin and cortisol levels were reduced. These effects were also evident 1 month post treatment and indicate khat may exert a transient effect on male fertility by interfering with the hormonal profiles.

J Reprod Immunol, 2006

Human leukocyte antigen-G (HLA-G) is a major histocompatibility complex class Ib gene expressed i... more Human leukocyte antigen-G (HLA-G) is a major histocompatibility complex class Ib gene expressed in normal organs and in some tumors. The glycoproteins encoded by this gene are best known for their immunosuppressive properties. Because isoform-specific expression of HLA-G in male reproductive organs has not been reported, we investigated HLA-G1, -G2, -G5, -G6 mRNAs and proteins in four-to-five samples of normal prostate glands, prostates with benign prostatic hyperplasia and prostate adenocarcinomas using RT-PCR and immunohistochemistry. All tissues contained HLA-G1, -G2, -G5 and -G6 specific mRNAs, but only HLA-G5 protein was detectable. In normal prostate glands, HLA-G5 protein was prominent in the cytoplasm of tubuloglandular epithelia and in glandular secretions. Staining was reduced in samples of benign prostatic hyperplasia but remained localized to the cytoplasm of glandular epithelia and secretions. In prostatic adenocarcinomas, HLA-G5 protein was detectable mainly in the secretions. Thus, HLA-G5 but not HLA-G1, -G2 or -G6 is produced in the normal prostate and is present in prostatic secretions. In addition, normal cellular localization is disturbed in benign and malignant prostatic adenocarcinomas. The results are consistent with this molecule may influencing female immune receptivity to sperm and suggest that such immunosuppression could be disturbed in men with prostatic adenocarcinomas.

Reproductive biology and endocrinology : RB&E, 2006

Pregnancy in mammals featuring hemochorial placentation introduces a major conflict with the moth... more Pregnancy in mammals featuring hemochorial placentation introduces a major conflict with the mother's immune system, which is dedicated to repelling invaders bearing foreign DNA and RNA. Numerous and highly sophisticated strategies for preventing mothers from rejecting their genetically different fetus(es) have now been identified. These involve production of novel soluble and membrane-bound molecules by uterine and placental cells. In humans, the placenta-derived molecules include glycoproteins derived from the HLA class Ib gene, HLA-G. Isoforms of HLA-G saturate the maternal-fetal interface and circulate in mothers throughout pregnancy. Uteroplacental immune privilege for the fetus and its associated tissues is believed to result when immune cells encounter HLA-G. Unequivocally demonstration of this concept requires experiments in animal models. Both the monkey and the baboon express molecules that are similar but not identical to HLA-G, and may comprise suitable animal models...

Placenta, 2007

HLA-G is an HLA class Ib gene that is highly expressed in human trophoblast cells. The single HLA... more HLA-G is an HLA class Ib gene that is highly expressed in human trophoblast cells. The single HLA-G mRNA is alternatively spliced to generate at least seven transcripts, three of which encode soluble isoforms. Many studies have shown that high levels of soluble antigens are associated with successful implantation and graft acceptance. To study expression, regulation and functions of two of the soluble isoforms, HLA-G5 and HLA-G6, we generated recombinant proteins in eukaryotic cells and developed monoclonal antibodies specific for each of the two proteins. In addition, we investigated the olive baboon Paan-AG gene as a potential functional correlate of HLA-G. Here, we present summaries of the studies that have been conducted in our laboratory using these tools and discuss the results within the context of the research on this topic that is ongoing in ours and other laboratories worldwide. Collectively, the data indicate that soluble HLA-G is a critical contributor to immune privilege in pregnancy and imply that this placenta-derived substance may impact other pathways leading to successful reproduction.

Placenta, 2008

Human placentas are sources of cytokines, hormones and other substances that program receptive ce... more Human placentas are sources of cytokines, hormones and other substances that program receptive cells. One of these substances is HLA-G, which influences the functioning of both leukocytes and endothelial cells. In this study we investigated the possibility that these and/or other types of cells in extraembryonic fetal tissues might respond to HLA-G by interacting with one or another of the leukocyte immunoglobulin-like receptors (LILR). LILRB1 is expressed by most leukocytes and LILRB2 is expressed primarily by monocytes, macrophages and dendritic cells. Analysis of term placentas by immunohistochemistry and Real Time PCR demonstrated that LILRB1 and LILRB2 protein and specific messages are produced in the mesenchyme of term villous placenta but are differently localized. LILRB1 was abundant in stromal cells and LILRB2 was prominent perivascularly. Neither receptor was identified in trophoblast. Further investigation using double label immunofluorescence indicated that placental vascular smooth muscle but not endothelia exhibit LILRB2. Term umbilical cord exhibited the same LILRB2 patterns as term placenta. Samples obtained by laser capture dissection of vascular smooth muscle in umbilical cords demonstrated LILRB2 mRNA, and double labeling immunofluorescence showed that cord vascular smooth muscle but not endothelium exhibited LILRB2 protein. The presence of LILRB1 in placental stromal cells and LILRB2 in vascular smooth muscle strongly suggest that HLA-G has novel functions in these tissues that could include regulation of placental immunity as well as development and function of the extraembryonic vasculature.

Placenta and Trophoblast, 2005

A wide variety of techniques has been developed for qualitative and quantitative analysis of gene... more A wide variety of techniques has been developed for qualitative and quantitative analysis of gene expression in human cells and tissues. Two commonly used methods are reverse-transcription (RT)-polymerase chain reaction (PCR) to analyze the transcribed messenger RNAs (mRNA) and immunohistochemistry to detect the translated proteins. These techniques can be modified and adapted for use in analyzing gene expression in animal models. In particular, as a result of the close phylogenetic relationship between humans and nonhuman primates, human reagents, especially antibodies, cross-react with nonhuman primate tissues. However, the results are not always satisfactory as some antibodies may cross-react with irrelevant antigens in these tissues. In this chapter, we describe the use of RT-PCR and immunohistochemical techniques to analyze expression of Paan-AG, a novel class lb major histocompatibility complex antigen in the olive baboon (Papio anubis) placenta. We used Paan-AG-specific primers to amplify Paan-AG transcripts from baboon placenta, and generated Paan-AG isoform-specific polyclonal antibodies for use in immunohistochemistry.

Virology, 2006

Although it is now well established that a substantial proportion of wild-living primates in sub-... more Although it is now well established that a substantial proportion of wild-living primates in sub-Saharan Africa harbor SIV, no study to date has examined to what extent the various species are naturally infected. In this study, we first describe the development and validation of sensitive and specific SIV antibody detection assays representing all major known primate lentiviral lineages on a panel of 207 sera from 11 different primate species with known infection status. The newly developed assays were then used to determine SIV prevalence rates in nine primate species native to Cameroon. Analysis of 722 sera revealed widely varying prevalence rates, ranging from an apparent absence of SIV infection in crested mona (0/70), grey cheeked (0/36) and agile mangabeys (0/92), to prevalence rates of 3%, 4%, 11%, 27%, 39% and 52% for mustached (6/203), greater spot-nosed (8/193), northern talapoin (3/26), mantled guereza (14/52), De Brazza's (9/23) and mandrill (14/27) monkeys, respectively. The epidemiology of naturally occurring SIV infections is thus more complex than previously appreciated and the various non-human primate hosts seem to differ in their susceptibility to SIV infection. The newly developed assays should now permit to define with greater accuracy existing SIV reservoirs and associated human zoonotic risk. D

Journal of Virology, 2004

Africa have now been reported; yet, our understanding of the origins, evolutionary history, and g... more Africa have now been reported; yet, our understanding of the origins, evolutionary history, and geographic distribution of these viruses still remains fragmentary. Here, we report the molecular characterization of a lentivirus (SIVdeb) naturally infecting De Brazza's monkeys (Cercopithecus neglectus). Complete SIVdeb genomes (9,158 and 9,227 bp in length) were amplified from uncultured blood mononuclear cell DNA of two wild-caught De Brazza's monkeys from Cameroon. In addition, partial pol sequences (650 bp) were amplified from four offspring of De Brazza's monkeys originally caught in the wild in Uganda. Full-length (9,068 bp) and partial pol (650 bp) SIVsyk sequences were also amplified from Sykes's monkeys (Cercopithecus albogularis) from Kenya. Analysis of these sequences identified a new SIV clade (SIVdeb), which differed from previously characterized SIVs at 40 to 50% of sites in Pol protein sequences. The viruses most closely related to SIVdeb were SIVsyk and members of the SIVgsn/SIVmus/SIVmon group of viruses infecting greater spot-nosed monkeys (Cercopithecus nictitans), mustached monkeys (Cercopithecus cephus), and mona monkeys (Cercopithecus mona), respectively. In phylogenetic trees of concatenated protein sequences, SIVdeb, SIVsyk, and SIVgsn/SIVmus/SIVmon clustered together, and this relationship was highly significant in all major coding regions. Members of this virus group also shared the same number of cysteine residues in their extracellular envelope glycoprotein and a high-affinity AIP1 binding site (YPD/SL) in their p6 Gag protein, as well as a unique transactivation response element in their viral long terminal repeat; however, SIVdeb and SIVsyk, unlike SIVgsn, SIVmon, and SIVmus, did not encode a vpu gene. These data indicate that De Brazza's monkeys are naturally infected with SIVdeb, that this infection is prevalent in different areas of the species' habitat, and that geographically diverse SIVdeb strains cluster in a single virus group. The consistent clustering of SIVdeb with SIVsyk and the SIVmon/SIVmus/SIVgsn group also suggests that these viruses have evolved from a common ancestor that likely infected a Cercopithecus host in the distant past. The vpu gene appears to have been acquired by a subset of these Cercopithecus viruses after the divergence of SIVdeb and SIVsyk.

Journal of Reproductive Immunology, 1997

Journal of Reproductive Immunology, 2006

Journal of Reproductive Immunology, 1999

Electron microscopic studies have revealed the presence of endogenous retroviral (ERV) particles ... more Electron microscopic studies have revealed the presence of endogenous retroviral (ERV) particles in normal primate placental tissues. These particles have ultrastructural similarities to type C retroviral particles and are mainly associated with the trophoblast. In normal human placental tissues, they have antigenic similarity with exogenous retroviruses, such as the human immunodeficiency virus (HIV), and may have a role to play in the regulation of cellular gene expression, syncytiotrophoblast formation or pregnancy-related immunosuppression. In this study, a panel of antibodies (polyclonal and monoclonal antibodies) against viral proteins (anti-HIV and anti-SIV) and endogenous retroviral (ERV) proteins were assessed by immunohistochemistry and immunoblotting, for their cross-reactivity with ERV particles isolated from normal baboon placental tissues. The antibodies (anti-HERV-K RT, anti-ERV3 en6, anti-HIV-1 p17, anti-HIV-2 gp120) reacted positively with the syncytiotrophoblast and each antibody recognized one or two proteins of molecular weights (MW) 38, 58 or 64 kDa present in the baboon placental villous tissues and SIV-infected molt-4 Cl 8 cells, but not in uninfected cells. The results of this study confirm the specific expression of

Journal of Medical Primatology, 1998

Endogenous retroviral particles (ERVs) have been detected in the genome of all eukaryotes. They a... more Endogenous retroviral particles (ERVs) have been detected in the genome of all eukaryotes. They are generally non-pathogenic except in mice where they have been found to induce tumors and immunological disorders. The ERVs have morphological features consistent with type-C retroviral particles and are commonly expressed in normal placental villous tissues. ERVs may have a role in the regulation of placental gene expression, syncytiotrophoblast formation, or pregnancy-related immunosuppression. In this study, well-characterized antibodies (monoclonal and polyclonal antibodies) raised against retroviral proteins (anti-HIV and anti-SIV) and endogenous retroviral (ERV) particles were assessed for their cross-reactivity (by using immunohistochemistry) with normal baboon placental and other adult tissues. The monoclonal antibodies to exogenous retroviral proteins (anti-HIV-2 gp120, anti-HIV-1 gp41, anti-SIVmac p27, anti-HIV-1 RT, and anti-HIV-2 core protein) showed specific immunohistochemical reactivity with the syncytiotrophoblast. Antibodies to endogenous retroviral gene products (anti-ERV3 env, anti-HERV-K RT, and anti-HERV-K env) also reacted in a similar manner and did not cross-react with other adult tissues. These studies have shown that retroviral-cross-reactive proteins are expressed in baboon placental syncytiotrophoblast and may have a role to play at the feto-maternal interface.

Journal of Ethnopharmacology, 2006

Immunology, 2007

Human leucocyte antigen-G (HLA-G) is a natural immunosuppressant produced in human placentas that... more Human leucocyte antigen-G (HLA-G) is a natural immunosuppressant produced in human placentas that binds differently to the inhibitory leucocyte immunoglobulin-like receptors LILRB1 (ILT2) and LILRB2 (ILT4) according to its biochemical structure. To predict the binding functions of the HLA-G5 soluble isoform synthesized in placental villous cytotrophoblast (vCTB) cells, we investigated structural features of this protein. Biochemical and immunological studies showed that vCTB cell HLA-G5 heavy (H)-chain proteins are disulphide-bonded homodimers unassociated with beta(2)-microglobulin (beta2m) light-chain proteins. Although comparatively low levels of beta2m messenger RNA (mRNA) were identified by real-time reverse transcription-polymerase chain reaction, immunoprecipitation studies failed to detect beta2m protein even when specific mRNA was doubled by transduction of a lentivirus-beta2m complementary DNA into vCTB cells. No abnormalities were identified in the translational start codon of vCTB cell beta2m mRNA and differentiation into syncytium did not promote beta2m synthesis. The failure of vCTB cells to exhibit beta2m in vitro was paralleled by a lack of detectable beta2m in vCTB cells in vivo. Lack of the beta2m protein could be the result of low levels of beta2m transcripts or of as yet unidentified translational defects. Experiments with recombinant ectodomains of LILRB indicate that beta2m-free HLA-G binds strongly to LILRB2, a receptor that is expressed by macrophages. This potentially immunosuppressive cell type is abundant in the pregnant uterus. Thus, our findings are consistent with the postulate that the natural beta2m-free homodimeric form of HLA-G5 synthesized in primary vCTB cells could comprise a particularly effective tolerogenic molecule at the maternal-fetal interface.

Immunogenetics, 2007

The human leukocyte antigen-G (HLA-G) gene encodes a protein that is highly expressed at the huma... more The human leukocyte antigen-G (HLA-G) gene encodes a protein that is highly expressed at the human maternal-fetal interface during pregnancy and may be critical to the survival of the semiallogenic fetus. A unique feature of this gene is a 13-bp deletion in the proximal promoter that renders it unresponsive to transactivation by the nuclear factor-κB (NF-κB). We previously showed that the proximal promoter of Paan-AG, the functional homologue of HLA-G in the olive baboon (Papio anubis), is intact. We cloned the promoters of two putative Paan-AG alleles (AG1 and AG2) and identified a number of regulatory elements including two κB sites. In the current study, binding and activity of the two κB elements in each putative allele were assessed by electrophoretic mobility shift and supershift assays. Functional activity was determined using luciferase reporter assays. The κB1 and κB2 elements in AG1 bound NF-κB with similar affinity. In contrast, the κB1 element of AG2 bound NF-κB with a much higher affinity than AG-1 κB1 (a 30-fold increase), whereas κB2 did not bind. Mutagenesis analysis showed that the difference in binding intensities was due to two nucleotides in the 3′ end of κB1. Similarly, failure of AG2 κB2 binding was a result of the last nucleotide in the 3′ end that differed from the consensus; mutating this nucleotide to match the consensus reestablished binding. Functional activity of the two putative alleles also differed; AG1 luciferase activity was consistently lower than that of AG2. Mutating the last two nucleotides in the 3′ end of AG1 κB1 resulted in increased luciferase activity to levels comparable to that of AG2. Overall, these results show that in vitro variations in the promoter region may influence transcription of Paan-AG.

Immunogenetics, 2002

The human class Ib major histocompatibility complex (MHC) molecule, HLA-G, is unique in its limit... more The human class Ib major histocompatibility complex (MHC) molecule, HLA-G, is unique in its limited polymorphism, high expression in the placenta and generation of multiple transcripts by alternative splicing. The proteins encoded by these transcripts are believed to modulate maternal-fetal immunological relationships during pregnancy. The baboon placenta contains messages encoded by a novel MHC gene, Paan-AG, which is evolutionarily related to the HLA-A locus, but shares unique characteristics with HLA-G. In this study, we show that the Paan-AG message is alternatively spliced to generate at least seven transcripts. One of these transcripts retains intron 4 and encodes a soluble glycoprotein with three external domains and a unique 21-amino-acid sequence at the carboxyl terminus, similar to soluble HLA-G1. This glycoprotein was detected in first trimester placental villous cytotrophoblast and syncytiotrophoblast, and in extravillous cytotrophoblast cells in the basal plate in term placenta. Four of the transcripts (Paan-AG1, Paan-AG2, Paan-AG3, Paan-AG4) encode membranebound class Ib MHC glycoprotein isoforms. Paan-AG1 protein expression was similar to that of sPaan-AG, while Paan-AG2 protein was not detected in these tissues. The other two transcripts (Paan-AGx and Paan-AGxi) contain a truncated exon 3 and multiple stop codons. Paan-AG1 and Paan-AGx transcripts were detected in a number of non-placental tissues, but these transcripts contained multiple stop codons. Because of the structural similarities and common features of organspecific expression and splicing of the message, studies on Paan-AG may be of value in dissecting the functions of the class Ib proteins in human pregnancy.

Immunogenetics, 2004

Potential regulatory sequences in the untranslated regions of the baboon MHC class Ib gene, Paan-... more Potential regulatory sequences in the untranslated regions of the baboon MHC class Ib gene, Paan-AG, more closely resemble those in the human MHC class Ia genes than those in the class Ib gene, HLA-G Abstract The baboon major histocompatibility complex (MHC) class Ib gene, Paan-AG, is structurally similar to the human MHC class Ia gene, HLA-A, but exhibits characteristics similar to those of the class Ib gene HLA-G. These include limited polymorphism, alternative splicing of a single message, and restricted tissue distribution, with high expression in the placenta. In order to determine whether regulatory elements controlling expression of Paan-AG resemble those of HLA-A or HLA-G, we cloned the 5′ and 3′ untranslated regions of Paan-AG. Unexpectedly, sequence comparisons showed that potential regulatory elements in Paan-AG strikingly resembled those in HLA-A and differed in major respects from those in HLA-G. Unlike HLA-G, Paan-AG contained an intact interferon-γ stimulated response element (ISRE) in the promoter. Studies using luciferase reporter assays showed that the Paan-AG ISRE was functional. The basal activity of the Paan-AG ISRE and its response to interferon-γ was similar to that of class Ia MHC genes. Further, we identified an ISRE in the 3′ untranslated region of Paan-AG that is known to be functional in HLA-A2 but is deleted in HLA-G. These experiments predict that functional studies may demonstrate differences in regulation of expression of Paan-AG and HLA-G genes, which could restrict the use of the baboon as a primate model for studying HLA-G expression and function.

East African Medical Journal, 2006

Objective: To review recent research findings on the specific expression of endogenous retroviral... more Objective: To review recent research findings on the specific expression of endogenous retroviral sequences (ERVS) in reproductive tissues and their possible physiological roles. ERVS have been implicated in several biological events such as induction of resistance to exogenous retrovirus invasion, involvement in placental trophoblast formation, sperm maturation and differentiation; and stimulation of local immunosuppression to protect the foetus from immunological attack. Data sources: Critical review of relevant articles and abstracts cited in international and local journals, literature searches on Medline and Medchem up to 2005. Data synthesis: Retroviruses have been implicated in the induction of tumour and immunological disorders. Over the years, endogenous retroviruses (ERVs) and retroviral elements have been detected in the genome of many vertebrate species, including primates. The evidence for the presence of retroviruses in the primate tissues such as the placenta, ovary, breast, testis and epididymis has been documented using electron microscopic studies. Retrovirus-like particles were found budding from the basal membrane of syncytiotrophoblasts, as well as in tumour cell lines in embryonic carcinoma or teratocarcinomas. Apart from their pathological effects, recent evidence suggests that these ERVs may play useful roles in normal physiological events. Results: Recent studies indicate the expression of endogenous retroviruses in the testis, epididymis, placenta and breast. However, limited data exist on the detection of ERVs in the ovary. Overall, the precise functions for ERVs in these tissues are not well understood. In the testis and epididymis, speculative functions may include among others spermatogenesis and/or sperm maturation (differentiation) whereas in placenta they are possibly associated with trophoblast fusion and locally induced immunosuppression to protect the foetus from immunological attack. Experiments in our laboratory have indicated restricted expression of retroviral antigens including baboon endogenous retroviral proteins (BERV), ERV-3, HIV-1 gp41 and HERV-K env in the baboon ovary. Conclusion: ERVs are specifically expressed in different mammalian reproductive tissues and may have unique physiological roles.