Estrogen accelerates immune complex glomerulonephritis but ameliorates T cell-mediated vasculitis and sialadenitis in autoimmune MRL lpr/lpr mice.

Estrogen is known to influence immune responses in healthy subjects in a dichotomous fashion. Thus, in number of previous studies we and others have demonstrated that B cell activities are augmented after exposure to estrogen whereas T cell reactivity is suppressed. Furthermore, it has been shown that this hormone has significant impact on the course of certain human and experimental autoimmune diseases. In this study we report that treatment with physiological doses of estradiol exerts dichotomous effects on different manifestations of the lupus disease in MRL/l mice. On one hand immune complex-mediated glomerulonephritis was significantly accelerated. This outcome was due to polyclonal B cell activation with increased production of antibodies to double-stranded DNA and formation of circulating immune complexes. In contrast, T cell-mediated lesions such as focal sialadenitis, renal vasculitis, and periarticular inflammation were all significantly ameliorated in MRL/l mice exposed to estrogen. Thus, we were able to demonstrate that, within one subject and even within one organ, administration of estrogen leads to differential outcome of SLE morbidity. We propose that the differential effect of estrogen on the manifestations of the autoimmune disease of MRL/l mice is due to its dichotomous effects on B and T cell-mediated immune responses.     

Demonstration of immunoreactive sites on cartilage after in vivo administration of biotinylated anti-type II collagen antibodies

Administration of biotinylated monoclonal antibodies provides the basis of a simple technique for identifying immunoreactive sites in vivo. Biotinylated anti-type II collagen antibodies were injected intraperitoneally into normal DBA/1 mice. The mice were sacrificed after 96 hr and the front paws removed and decalcified to allow tissue sectioning before snap-freezing. Binding of antibodies in vivo was visualized with affinity cytochemical staining using avidin-biotin-peroxidase complexes. Specific binding of antibodies to cartilaginous structures was seen after injection of 20-500 micrograms biotinylated monoclonal or polyclonal anti-type II collagen antibodies, but not after injection of a biotinylated control antibody. This technique should further the detection and localization studies of tissue components involved in the dynamics of physiological and pathological processes.     

Origin of the autoreactive anti-type II collagen response. II. Specificities, antibody isotypes and usage of V gene families of anti-type II collagen B cells

Autoantibodies play an important role in the pathogenesis of type II collagen-induced arthritis in mice. We have earlier reported a high frequency of cells producing anti-CII autoantibodies and a low frequency of cells producing multispecific antibodies, in regional lymph nodes 9 to 11 days after primary immunization with CII. It is shown here that anti-CII antibodies produced during primary immune response are IgG-antibodies mainly of IgG2a, IgG1 and IgG2b subclasses while IgM antibodies dominate primary responses elicited by OVA and denatured CII as analyzed with a large panel of hybridomas. Anti-CII antibodies generated during the primary response recognize at least five different epitopes on the CII molecule. The specificities of these antibodies for various epitopes result from combinational association of products encoded by genes derived from various VH and VK families and/or by the occurrence of somatic mutations. It is suggested that the primary anti-CII autoantibody response involves activation of memory B cells and is in this aspect different from the origin of "natural" autoantibodies.     

A Case of Neospora Associated Bovine Abortion in Sweden

Neospora caninum is a recently recognized protozoan organism that causes fatal neuromuscular disease in dogs and abortions and stillbirths in cattle and other animals (Dubey & Lindsay 1993). The parasite is morphologically similar and phylogenetically very closely related to the cyst-forming coccidium Toxoplasma gondii (Ellis et al. 1994, Holmdahl et al. 1994). This group of parasites has a two-host life cycle principally involving a carnivorous definitive host and a herbivorous or omnivorous intermediate host. However, with N. caninum, there is as yet no knowledge of any definitive host harbouring sexual stages of the parasite. The only known route of transmission is vertical from mother to foetus (Dubey & Lindsay 1993).     

Synthesis of fused bicyclic thioglycosides of N-acylated glucosamine as analogues of mycothiol

The synthesis of a fused bicyclic thioglycoside analogue of mycothiol, (3R)-3-acetylamino-4-one-6,7-dihydro-(1',2'-dideoxy-beta-D-glucopyranoso)[2',1'-f]-1,5-thiazepane (5), is reported. Treatment of phthalimido-protected peracetylated glucosamine with N-acetyl-cysteine and boron trifluoride-etherate gave the beta-linked thioglycoside, which was deprotected and cyclized, using HOBt and EDCl to form the lactam and giving the target structure. This mycothiol mimic and its tri-O-acetate will be investigated as potential inhibitors of enzymes involved in the biosynthesis of mycothiol. The protected derivative also has the potential to be an alpha-selective N-cysteinyl glucosamine donor; however, initial glycosylation attempts failed due to the apparent stability of the fused bicyclic system.     

IFN-beta gene deletion leads to augmented and chronic demyelinating experimental autoimmune encephalomyelitis

Since the basic mechanisms behind the beneficial effects of IFN-beta in multiple sclerosis (MS) patients are still obscure, here we have investigated the effects of IFN-beta gene disruption on the commonly used animal model for MS, experimental autoimmune encephalomyelitis (EAE). We show that IFN-beta knockout (KO) mice are more susceptible to EAE than their wild-type (wt) littermates; they develop more severe and chronic neurological symptoms with more extensive CNS inflammation and demyelination. However, there was no discrepancy observed between wt and KO mice regarding the capacity of T cells to proliferate or produce IFN-gamma in response to recall Ag. Consequently, we addressed the effect of IFN-beta on encephalitogenic T cell development and the disease initiation phase by passive transfer of autoreactive T cells from KO or wt littermates to both groups of mice. Interestingly, IFN-beta KO mice acquired a higher incidence and augmented EAE regardless of the source of T cells. This shows that the anti-inflammatory effect of endogenous IFN-beta is predominantly exerted on the effector phase of the disease. Histopathological investigations of CNS in the effector phase revealed an extensive microglia activation and TNF-alpha production in IFN-beta KO mice; this was virtually absent in wt littermates. This coincided with an increase in effector functions of T cells in IFN-beta KO mice, as measured by IFN-gamma and IL-4 production. We suggest that lack of endogenous IFN-beta in CNS leads to augmented microglia activation, resulting in a sustained inflammation, cytokine production, and tissue damage with consequent chronic neurological deficits.     

Antibiotic-free bacterial strain selection using antisense peptide nucleic acid

Antibiotics are widely useful in medicine, agriculture, and industrial fermentations. However, increasing problems with resistant strains call for restrained use and alternative strategies. Antisense peptide nucleic acids (PNAs) show potent bactericidal effects when targeted against the essential Escherichia coli acpP gene. Aside from attractive antimicrobial therapeutic possibilities for such antisense PNAs, we considered that they could be used as a substitute for antibiotics in bacterial strain selection. Here, treatment of a mixture of E. coli wild-type cells and cells carrying a binding-site altered copy of acpP (acpP-1) with anti-acpP PNA completely killed wild-type cells within 2 h, whereas cells carrying acpP-1 proliferated. Furthermore, electrotransformation of E. coli cells with the plasmid carrying acpP-1 followed by PNA selection gave rise to only true transformants. Unlike previous antibiotic-free selection strategies, this procedure does not require special growth environments or special host strains. Also, the PNA-selected cells grow at a near normal rate. The results open possibilities to use antisense PNAs for strain selection and construction in research and industrial application.     

The translation start codon region is sensitive to antisense PNA inhibition in Escherichia coli

Antisense peptide nucleic acids (PNA) can inhibit bacterial gene expression with gene and sequence specificity. Using attached carrier peptides that aid cell permeation, the antisense effects when targeting essential genes are sufficient to prevent growth and even kill bacteria. However, many design uncertainties remain, including the difficult question of target sequence selection. In this study, we synthesized 90 antisense peptide-PNAs to target sequences in a head to tail manner across the entire length of the mRNA encoding beta-lactamase. The results from this scan pointed to the start codon region as most sensitive to inhibition. To confirm and refine the result, a higher-resolution scan was conducted over the start codon region of the beta-lactamase gene and the essential Escherichia coli acpP gene. For both genes, the start codon region, including the Shine-Dalgarno motif, was sensitive, whereas antisense agents targeted outside of this region were largely ineffective. These results are in accord with natural antisense mechanisms, which typically hinder the start codon region, and the sensitivity of this region should hold true for most bacterial genes as well as for other RNase H-independent antisense agents that rely on a steric blocking mechanism. Therefore, although other design parameters are also important, the start codon region in E. coli mRNA is the most reliable target site for antisense PNAs.     

Fragmentation of two quantitative trait loci controlling collagen-induced arthritis reveals a new set of interacting subloci

Linkage analysis of F(2) crosses has led to identification of large numbers of quantitative trait loci (QTL) for complex diseases, but identification of the underlying genes has been more difficult. Reasons for this could be complications that arise from separation of interacting or neighboring loci. We made a partial advanced intercross (PAI) to characterize and fine-map linkage to collagen-induced arthritis in two chromosomal regions derived from the DBA/1 strain and crossed into the B10.Q strain: Cia7 on chromosome 7 and a locus on chromosome 15. Only Cia7 was detected by a previous F(2) cross. Linkage analysis of the PAI revealed a different linkage pattern than the F(2) cross, adding multiple loci and strong linkage to the previously unlinked chromosome 15 region. Subcongenic strains derived from animals in the PAI confirmed the loci and revealed additional subloci. In total, no less than seven new loci were identified. Several loci interacted and three loci were protective, thus partly balancing the effect of the disease-promoting loci. Our results indicate that F(2) crosses do not reveal the full complexity of identified QTLs, and that detection is more dependent on the genetic context of a QTL than the potential effect of the underlying gene.