James W. Larrick

Cellular and molecular regulation of tumor necrosis factor-alpha production by pentoxofylline

Tumor necrosis factor-alpha (TNF), a mononuclear phagocyte (MO)-derived peptide, is increasingly being recognized for its pleomorphic immunologic effects. A number of investigations have demonstrated that lipopolysaccharide (LPS) can induce TNF synthesis, yet mechanisms that regulate TNF expression at the cellular and molecular levels have not been fully elucidated. In this study, we present data demonstrating pentoxifylline, a methylxanthine, is efficacious in suppressing LPS-induced MO-derived TNF at the level of both TNF mRNA accumulation and TNF supernatant bioactivity. Pentoxifylline, at a dose of 1 x 10-5M, suppressed the production of both biologically active TNF and TNF mRNA expression by more than 50%. Furthermore, additional methylxanthines and dibutyryl cAMP have similar effects on TNF expression. These data support the mechanism for this suppressive effect is via the generation of intracellular cAMP.

Message amplification phenotyping (MAPPing)--principles, practice and potential.

Message amplification phenotyping (MAPPing) is a sensitive polymerase chain reaction (PCR)-based technique for the rapid determination of the mRNA phenotype of small numbers of cells. Isolated mRNA is reverse-transcribed (RT) into DNA, and then the DNA fragments corresponding to proteins or genes of interest are specifically amplified by PCR. The technique, also called RT-PCR, has wide application in biology and medicine, and comparison with bioassays demonstrates that MAPPing saves significant time and material. The technique should be applicable to analyse mRNAs of virtually any tissue or cell type.

Systematic generation of antigen specific human monoclonal antibodies with therapeutical activities using active immunization

Presenting a panel of human hybridomas secreting serospecific antibodies which confer a high degree of protection against fatal infection with Pseudomonas aeruginosa, we report an efficient approach for a systematic generation of antigen specific human monoclonal antibodies with biological activity. This approach is based on active immunization and antigen specific panning. Individuals were immunized with polysaccharides isolated from LPS of Pseudomonas aeruginosa conjugated to toxin A. Specific B cells were isolated and enriched by panning of blood samples taken at the time point with the highest frequency of fuseable cells. These cells were then transformed with Epstein-Barr virus. Arising lymphoblastoid cell lines were screened for the secretion of anti-LPS antibodies by enzyme-linked immunosorbent assay and fused to a murine-human heteromyeloma cell line. Hybridomas were selected for high levels of antibody secretion and binding to intact bacteria as determined by an immunofluorescence microscopy assay. The observation that protective capacity of an antibody was associated with its ability to bind to LPS determinants accessible on the bacterial cell surface allowed for an effective screening for therapeutically interesting human monoclonal antibodies. Out of four immunized individuals, 15 lymphoblastoid cell lines with anti-LPS activity could be isolated, and 8 hybridomas, which cover the majority of the common Pseudomonas aeruginosa serotypes, were characterized further. The generation of monoclonal anti-Pseudomonas aeruginosa toxin A, anti-Klebsiella capsular polysaccharides, and anti-Escherichia coli LPS antibodies suggests that the success of this approach is not limited to the generation of human monoclonal antibodies of a particular specificity or to the use of antigens of a particular character.

Human monoclonal antibody against group A red blood cells

Abstract. A human monoclonal antibody with anti‐A specificity was generated by Epstein‐Barr virus transformation of lymphocytes isolated from splenic tissue after in vitro stimulation with group A red blood cells. This antibody is of the IgM class and directly agglutinates group A red blood cells. Type A1, A2, Aint, A3, Ax, Aend and A5 cells were agglutinated by the reagent indicating a single determinant is shared by these A subgroups.

In Vitro Expansion of Human B Cells for the Production of Human Monoclonal Antibodies

Early work from a number of laboratories has demonstrated the low frequency with which peripheral human B cells secreting specific monoclonal antibodies can be rescued by cell fusion (Olsson et al., 1983; Kozbor and Roder, 1983b, 1984). Use of spleen and lymph node tissue only marginally improved these results. The development of new human fusion partners (Larrick et al.,1983; Glassy et al., 1983; Kozbor and Roder, 1983; Chiorazzi et al., 1982; Abrams et al., 1983), mouse—human heteromyeloma cell lines (Teng et al., 1983), mouse—human fusion partners (Kozbor and Roder, 1984, 1983b), and optimization of fusion protocols (Buck et al., 1984; Truitt et al., 1984) has slightly increased the frequency of specific antibodies, but the efficiency is still far less than that of immunized mice or rats. Even with a greater understanding of the postimmunization kinetics of immune B-cell precursors in peripheral human blood (Callard et al., 1982; Butler et al.,1983; Astaldi et al., 1982), production of antibody-producing human hybridomas is still an uncommon event.

Digital and palmar dermatoglyphic patterns among natives of extreme northwest Nepal.

A dermatoglyphic study was made of two high-altitude populations inhabiting the same ecozone in extreme northwest Nepal. Despite cultural and linguistic differences there appears to be a significant amount of dermatoglyphic homology between the Buddhist-Tibetan villages and their Hindu-Caucasian neighbors.