We have generated a monoclonal B-cell mouse by introducing homozygous, nonfunctional
RAG-2 alleles and a &#x3BB;1 light-chain transgene into the quasi-monoclonal (QM) mouse, which
contains a &#x201C;knocked-in&#x201D; VHDJH rearrangement. Thus, this mouse, which we call MonoB, is
devoid of T cells and contains preformed heavy- and light-chain genes encoding immunoglobulin
with an anti-NP specificity. The MonoB mouse allows us to examine immunoglobulin
diversity in the absence of processes mediated by V(D)J recombination and T cells. Here we
report that not only is the MonoB&#x27;s primary immunoglobulin repertoire monoclonal, but also
that its secondary repertoire is not further diversified by V-gene replacement or gene conversion.
Among 99 heavy-chain and 41 &#x3BB; light-chain genes from peripheral B cells of the
MonoB mouse, there were no V-gene replacements. When compared to the QM mouse,
which has RAG activity, and for which V-gene replacement is the major diversifying mechanism,
these data suggest that V-gene replacement is mediated by V(D)J recombination and
not by other recombination systems.

Marilia Cascalho

Denise A. Martin

Jamie Wong

Queenie Lam

Matthias Wabl

Gillian E. Wu

A Mouse with a Monoclonal Primary lmmunoglobulin
Repertoire not Further Diversified by V-Gene
Replacement

Female reproduction is the only system subjected to well defined periodic changes. The final
stage of the menstrual cycle in mammals is the maturation of the ovum and the preparation of
the female organism to support fetal development fertilization. Once pregnancy occurs, both
maternal and fetal sites emit regulatory signals to ensure embryo development and maternal
protection against a graft versus host (GvH) reaction initiated by the semi-allogeneic fetus.
We and others have previously shown that each day of fetal development in mice is characterized
by different cytokine production, detected not only at the proximity of the feto-placental
unit (decidua, uterus), but also in maternal lymphoid organs (spleen), as well as in the
serum. In the present study, we concentrated on the menstrual cycle and the preimplantation
stages of pregnancy and defined the levels of GM-CSF, IL-10, IL-6, and IL-3 in the murine
uterus during anoestrus, proestrus, oestrus, and second and third day of gestation. We show by
immunofluorescence and ELISA techniques that GM-CSF is maintained at high levels during
anoestrus, proestus, oestrus, and the second day of pregnancy while dropping on the third day.
IL-3 levels are found elevated during proestrus, second and third day of gestation, IL-6
increases essentially during proestrus, whereas the production of IL-10 was detected during
oestrus and the early stages of pregnancy. Immunoperoxidase staining on frozen sections of
uteri during the early gestational period localize GM-CSF and IL-3 production in the
endometrium, IL-10 in the endometrium on the second day of pregnancy, and
endometrium/myometrium on the third day. Low levels of IL-6 could be detected in the
endometrium/epithelium on the second day and endometrium/myometrium on the third day
of gestation. The role of IL-3, IL-10, and, to a lesser degree, IL-6 is fortified by the embryo
itself, since these cytokines were found to be produced by blastocysts as well. These results
demonstrate the existence of a specific distribution of lymphokines within the uterine tissue,
the role of which is being discussed.

Irene Athanassakis

Vagia Farmakiotis

Lina Papadimitriou

Uterine Cytokine Production During the Menstrual Cycle and Preimplantation Stages in Mice

We have reinvestigated an important issue in the amphibian immunology that has not beensettled for years since the pioneer work of Triplett, concerning the necessity of being exposedto organ-specific antigens early in development. It was found that syngeneic lenses wererejected by frogs, Xenopus laevis, that had been enucleated (eye removed) during early larvallife. This rejection did not occur in intact frogs or in those enucleated in later larval or adultlife. Whereas the splenocytes from intact frogs did not proliferate in response to a co-culturedsyngeneic lens, those from frogs that had been enucleated at any of the larval stages, or evenafter metamorphosis, proliferated intensely. Both of these responses were shown to be thymus-dependent. In conclusion, it was demonstrated that the frog immune system rejectedeven syngeheic lenses by enucleation in early larval life and that it began to recognize thesyngeneic lenses by lymphoid proliferation after enucleation, even in later life.

Takeshi  Enomoto

Shin  Tochinai

Immune Response to &#8220;Self&#8221; Lens in Xenopus laevisEnucleated during Larval Life

Interleukin-12 (IL-12) is an important cytokine for maintainence of normal systemic defenseand bioregulation. The Japanese herbal medicine Sho-saiko-to (TJ-9) has been administeredto 1.5 million Japanese patients with chronic liver diseases. TJ-9 is known to significantlysuppress cancer development in the liver and has macrobiotic effects. In the present study, weexamined the in vitro production of IL-12 by circulating mononuclear cells from liver cirrhosispatients and the effects of TJ-9 on IL-12 production.The monocyte/macrophage fraction and the lymphocyte fraction of peripheral blood wereobtained from 11 HCV-positive liver cirrhosis patients and 12 healthy subjects. Interleukin-12levels in the supernatants were measured using ELISA kits. The levels of IL-12 produced bythe patients fractions were significantly lower than those produced by healthy subjects (p &#60;0.01, p &#60; 0.05). However, when TJ-9 was added to the cultures, the IL-12 production levels inboth cell fractions increased approximately three fold, and the levels from the monocyte/macrophagefraction were almost the same as those from healthy subjects. This effect of TJ-9 wasattributable to two of its seven herb components, that is, scutellaria root and glycyrrhiza root.One possible mechanism for the macrobiotic effects of TJ-9 on liver cirrhosis patients may bethe improvement in IL-12 production.

Masayoshi  Yamashiki

Akira  Nishimura

Xian-Xi  Huang

Tsutomu  Nobori

Seigo  Sakaguchi

Hiroyuki  Suzuki

Effects of the Japanese Herbal Medicine &#8220;Sho-saiko-to&#8221;(TJ-9) on Interleukin-12 Production in Patients withHCV-Positive Liver Cirrhosis

We have previously reported that reading-frame usage and functional diversification is developmentallyregulated, with virtually all TCRB DJ mRNA transcripts using a single readingframe at 8 weeks of gestational age, tapering to 50&#37; by adult life. We used the polymerasechain reaction to create genomic libraries of DJ rearrangements in the TCRB locus from thymusesat 7.7, 10, and 16 weeks of gestational age, and from adult thymuses. Clones were randomlypicked and sequenced to determine junctional sequences and reading-frameutilization. The resulting data address the hypothesis that cells bearing genomic joints in readingframe one are preferentially selected during fetal life. This hypothesis predicts that reading-frame bias would also be observed among genomic DJ joints. Instead, we observedrandom utilization of the three possible D-region reading frames among genomic D1s1 &#61;&#62;J1s1 joints during fetal life. Similar results were obtained at 7.7 weeks of gestational age in asecond thymus in which both RNA and DNA were simultaneously isolated and used to createlibraries of TCRBDJ transcripts or rearrangements. We conclude that reading-frame utilizationis random among genomic D1s1-JB1s1 rearrangements and that the preferential usage ofa single reading frame among mRNA transcripts of TCRB DJ transcripts is the result of preferentialtranscription of genomic TCRB DJ joints in a single reading frame, or that TCRB DJtranscripts have a longer half-life than transcripts in reading frames two or three.

James F. George

Yasushi  Matsuura

Jacquelyn A. Byrne

Eugene L. Liu

Denise R. Shaw

John F. Kearney

A Developmental Bias in Reading Frame Usage by HumanFetal Thymic TCRBDJ Transcripts is not Present inGenomic TCRBDJ Rearrangements

During embryogenesis in mouse, the thymus is seeded by waves of hematopoietic stem cells
that provide the first peripheral T lymphocytes after birth. It is known that embryo thymocytes
and adult thymocytes have different phenotypic and functional features. The identification
of genes expressed in the thymus only during embryogenesis would help to understand
the molecular basis underlying these characteristics. We used the mRNA differential display
technique to compare gene expression between thymus and kidney from embryo (171/2 days)
and adult mice. This technique is the method of choice for comparing gene expression
because it is able to display rapidly and simultaneously the mRNA complement from several
different types of cells. The major drawback of the method is that it leads to the cloning of
many false positives and therefore needs a high throughput method to screen for the truly differentially
expressed cDNAs. We combined advantages from previously described methods
in order to develop a new version of the mRNA differential display technique that is fast,
cheap, and reliable. Instead of oligo dT priming, we used random hexameres for the reverse
transcription of total RNA and 10-mer primers for the amplification of internal parts of the
cDNAs. We obtained reproducible and clean patterns of discrete bands. We were able to easily
identify DNAs differentially amplified between embryo and adult tissues (embryo specific;
E 58.73), between thymus and kidney (thymus specific; Thy 52.54), or between embryo
and adult thymus (embryo thymus specific; E Thy 58.73) cDNA fragments. After reamplification,
cloning, and sequencing of these DNA fragments, it appeared that in most cases, one
band corresponded to a single DNA sequence. On a northern blot, each of these candidate
genes recognized a transcript that is differentially expressed as expected. Thus, we report an
optimized, reproducible, and fast mRNA differential display method that overcomes the usual
problems met with the originally described technique or its reported modifications.

Steve Pascolo

Debbie Tsoukatou

Clio Mamalaki

Identification of Thymus Specific and Developmentally
Regulated Genes by an Improved Version of the mRNA
Differential Display Technique

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Journal of Immunology Research - Table of contents 1999

title

Journal of Immunology Research provides a platform for scientists and clinicians working in different and diverse areas of immunology and therapy.

Journal of Immunology Research

Table of Contents

Table of Contents: 1999

A Mouse with a Monoclonal Primary lmmunoglobulin Repertoire not Further Diversified by V-Gene Replacement

Uterine Cytokine Production During the Menstrual Cycle and Preimplantation Stages in Mice

Immune Response to “Self” Lens in Xenopus laevis Enucleated during Larval Life

Effects of the Japanese Herbal Medicine “Sho-saiko-to” (TJ-9) on Interleukin-12 Production in Patients with HCV-Positive Liver Cirrhosis

A Developmental Bias in Reading Frame Usage by Human Fetal Thymic TCRBDJ Transcripts is not Present in Genomic TCRBDJ Rearrangements

Identification of Thymus Specific and Developmentally Regulated Genes by an Improved Version of the mRNA Differential Display Technique