Richard L. Miller

Human TLR-7-, -8-, and -9-Mediated Induction of IFN-α/β and -λ Is IRAK-4 Dependent and Redundant for Protective Immunity to Viruses

n Summaryn n Five TLRs are thought to play an important role in antiviral immunity, sensing viral products and inducing IFN-α/β and -λ. Surprisingly, patients with a defect of IRAK-4, a critical kinase downstream from TLRs, are resistant to common viruses. We show here that IFN-α/β and -λ induction via TLR-7, TLR-8, and TLR-9 was abolished in IRAK-4-deficient blood cells. In contrast, IFN-α/β and -λ were induced normally by TLR-3 and TLR-4 agonists. Moreover, IFN-β and -λ were normally induced by TLR-3 agonists and viruses in IRAK-4-deficient fibroblasts. We further show that IFN-α/β and -λ production in response to 9 of 11 viruses tested was normal or weakly affected in IRAK-4-deficient blood cells. Thus, IRAK-4-deficient patients may control viral infections by TLR-3- and TLR-4-dependent and/or TLR-independent production of IFNs. The TLR-7-, TLR-8-, and TLR-9-dependent induction of IFN-α/β and -λ is strictly IRAK-4 dependent and paradoxically redundant for protective immunity to most viruses in humans.n n

A Fast Procedure for the Detection of Defects in Toll-like Receptor Signaling

OBJECTIVES. Inborn defects in Toll-like receptor signaling are recently described primary immunodeficiencies that predispose affected children to life-threatening infections. Patients with interleukin-1 receptor-associated kinase-4 deficiency are prone to invasive pneumococcal disease, and patients with UNC-93B deficiency are prone to herpes simplex virus encephalitis. These genetic disorders are underdiagnosed, partly because diagnosis currently requires expensive and time-consuming techniques available at only a few specialized centers worldwide. We, therefore, aimed to develop a cheap and fast test for the detection of defects in Toll-like receptor signaling. PATIENTS AND METHODS. We used flow cytometry to evaluate the cleavage of membrane-bound L-selectin on granulocytes in 38 healthy controls and in 7 patients with genetically defined Toll-like receptor signaling defects (5 patients with interleukin-1 receptor-associated kinase-4 deficiency and 2 patients with UNC-93B deficiency), on activation with various Toll-like receptor agonists. RESULTS. Impaired L-selectin shedding was observed with granulocytes from all of the interleukin-1 receptor-associated kinase-4-deficient patients on activation with agonists of Toll-like receptors 1/2, 2/6, 4, 7, and 8 and with granulocytes from all of the UNC-93B-deficient patients on activation with agonists of Toll-like receptors 7 and 8. All of the healthy controls responded to these stimuli. CONCLUSIONS. The assessment of membrane-bound L-selectin cleavage on granulocytes by flow cytometry may prove useful for the detection of primary immunodeficiencies in the Toll-like receptor pathway, such as interleukin-1 receptor-associated kinase-4 deficiency and UNC-93B deficiency. This procedure is cheap and rapid. It may, therefore, be suitable for routine testing worldwide in children with invasive pneumococcal disease and in patients with herpes simplex encephalitis.

SPECIFICITY OF SPERM CHEMOTAXIS AMONG GREAT BARRIER REEF SHALLOW-WATER HOLOTHURIANS AND OPHIUROIDS

Sperm chemotaxis and sperm motility activation by egg or ovarian extracts were demonstrated in 24 holothurians (six genera from three families) and 22 ophiuroids (eight genera from five families) from the Australian Great Barrier Reef. Specificity was observed mainly at the family level in holothurians, but a single case of specificity at the species level was found in the genus Bohadschia. No recognizable specificity was found between any of the currently recognized subgeneric groups within the genus Holothuria. A similar pattern of specificity was previously observed in a group of dendrochiridotid holothurians in temperate waters. n n n nIn contrast to the holothurians, specificity in ophiuroids existed mainly at the genus or species level. The best case of specificity at the species level was among six species in the genus Macrophiothrix which were reciprocally tested and a further three species in the same genus which were partially tested. Examples of partial specificity at the species level were also found in the general Ophiarthrum and Ophiocoma. The sperm chemotaxis assay reliably sorted to species a random collection of unidentified Macrophiothrix ophiuroids. These results suggest that sperm chemotaxis may play a role in gamete recognition prior to fertilization in one group of echinoderms. J. Exp. Zool. 279:189-200, 1997.

Some Observations on Sexual Reproduction in Tubularia

Observations of the sexual reproduction of the hydroid Tubularia have been made over a period of several years in connection with my work on animal sperm chemotaxis prior to fertilization. What follows is a summary of my findings, in an attempt to add this data to the published literature on an organism which has been used almost solely for studies of asexual reproduction (Rose, 1974).

Chemosensory phenomena during sexual interactions in gelatinous zooplankton

Gelatinous Zooplankton reproduce in an environment inimical to fertilization success because of the high potential for gamete dilution and the often wide dispersion of reproductive adults. Organisms in this group that shed sperm externally but fertilize internally face additional barriers to high fertilization success. Special reproductive adaptations probably exist to counter this problem. However, most of the inferences made about fertilization success in marine organisms are based on results using benthic species, gelatinous Zooplankton being very difficult to capture and maintain in sexual condition. As a result, very little has been done to determine what sort of adaptations to enhance fertilization rates exist in gelatinous species. Furthermore, there is little direct evidence supporting mutual approach of the sexes that would bring the ripe gametes closer together. Close approach may be accomplished by a number of physical agencies, however, and possibly by gamete adaptations such as long‐lived spe...

Evidence of dioecy in the mesopelagic ctenophore Bathocyroe fosteri (Lobata Ctenophora)

Summary Study of gonads of the mesopelagic ctenophore Bathocyroe fosteri revealed that the animals are dioecious for at least part of their life span. Specimens were collected using the Johnson Sea-Link I and II submersible vehicles during the periods of October 4–11, 1988, off the Bahamas Banks and July 18–20, 1991, off Cape Hatteras at depths of 550–900 m. Each animal was provisionally sexed by scanning all comb rows with a dissecting microscope. The provisional sex was confirmed after preparation of squashes of ad-tentacular and ab-tentacular comb rows for eggs and sperm at various magnifications using dark field optics and histological sections. With two exceptions, only one type of gamete was found in any sexual specimen and all comb rows bore the same type of gamete on both sides of the gastrovascular canal. Unlike Ocyropsis, the only other dioecious, planktonic ctenophore known, Bathocyroe has a gonad location typical of all other ctenophores. Our observations suggest that ctenophore dioecy is not restricted to the epipelagic, oceanic habitat and is not a property of a single genus with atypically located gonads. Dioecy has apparently evolved secondarily at least twice in habitats largely occupied by a group of related ctenophores which are all simultaneous hermaphrodites.

Norzooanemonin in the hydroidTubularia larynx

The hydrochloride of 1,3-dimethyl imidazole-4-carboxylic acid (norzooanemonin), has been isolated from the hydroidTubularia larynx and its structure determined by X-ray analysis.

Gamete interactions and fertilization behavior in the larvacean, Oikopleura dioica

Summary Oikopleura dioica are often cultured in the laboratory but for several reasons are clearly not in conditions similar to those of the open sea. Despite this impediment, some interesting behaviors of the spawning of adults and gametes in 3-ml well plates were observed from April and May 1992–1994 using the circulation devices of Fenaux and Cie and Gorsky and Fenaux (2002). Testis sac size measurements and observations of swimming behavior of numerous individual males were made during sperm release. We calculated the intervals between initiation of sperm release and its cessation, the sinking rates of individual eggs, the potential for re-fertilization of unfertilized eggs up to 24 h post release, and the effects of sperm concentration on fertilization success. We found that in a group of 74 males the total minutes of spawning time was 1,076 min with an average time to completion of spawning by all males at 14.54 min. Individual eggs sink in a 10-ml graduated cylinder at the rate of 2.35 mm/s in still water at 16°C. During sperm shedding, most males made a series of from one to five small horizontal loops every few seconds along the upper edge of a 3-ml well. The number of loops made increased during spawning. We suggest that this behavior is a decreasing regular spiral. Most males make clockwise loops while swimming against the inner edge of the wells but a few individuals rotated in the counterclockwise direction. Sperm aging was studied using sperm 100 min or older prior to the addition of newly spawned eggs. After fresh sperm were added to small groups of 24-h-old eggs incubated in 3-ml wells, several eggs cleaved properly and developed to the twitching larval stage. Fertilization success curves obtained on three different days using separate batches of eggs and sperm from each male produced nearly identical curves. However, sperm left standing at 16°C for longer than 100 min rapidly lost potency. Addition of higher sperm concentrations dramatically increased the fertilization rate.

Isolation of trimethyl amine oxide and other bases from the hydroid Tubularia larynx

1. Trimethyl amine oxide, dimethyl amine and choline chloride have been isolated from the marine hydroid Tubularia larynx and identified using physical constants and spectral data. 2. The presence of two guanidine molecules has been demonstrated.