Background-Upregulation of Th1 associated intrahepatic cytokines in chronic hepatitis C virus (HCV) infection should lead to a significant non-specific cellular immune response, a prerequisite for viral clearance. However, to date, the role of this non-specific response in HCV has been understudied. Aims-To analyse the intrahepatic macrophage activity in chronic HCV infection by immunostaining and by quantitation of cytokine mRNA. Methods-HCV positive liver tissues (chronic hepatitis, n=10; cirrhosis, n=5) were immunostained for CD68, MAC387, and semiquantitated by polymerase chain reaction for intrahepatic cytokine mRNAs (interferon (IFN ), interleukin 1 (IL-1 ), IL-6, IL-18, tumour necrosis factor (TNF ), and macrophage inflammatory protein 1 (MIP1 )). HCV negative normal liver tissues (for cytokines, n=6; for immunostaining, n=5) were included as controls. Results-MAC387+ cells were focally increased in areas of erosion at the limiting plate while lobular staining was minimal. CD68 + staining was diVuse in both portal (increased in HCV) and lobular areas. The portal tract (mean) density of CD68 + and MAC387 + cells was significantly increased in patients with HCV compared with normal tissue. IFN and IL-18 mRNA levels were highly correlated and significantly upregulated in chronic hepatitis and cirrhotic tissue versus controls. TNF mRNA was upregulated in chronic hepatitis without cirrhosis, while IL-6 mRNA was significantly downregulated. IL-1 , IL-6, and MIP1 mRNA levels were significantly correlated with portal tract MAC387 + cell density. Conclusions-The significant upregulation of IFN and IL-18 mRNA and significant correlations between IFN and other proinflammatory cytokines, suggest a Th1/cell mediated intrahepatic immune response in chronic HCV infection. However, further clarification of the cellular sources of these cytokines is required. (Gut 2000;46:260-269)

We have determined a critical period for vestibular development in zebrafish by using a bioreactor designed by NASA to simulate microgravity for cells in culture. A critical period is defined as the briefest period of time during development when stimulus deprivation results in long lasting or permanent sensory deficits. Zebrafish eggs were collected within 3 hours of being laid and fertilized. In experiment 1, eggs were placed in the bioreactor at 3, 24, 30, 36, 48, or 72 hours postfertilization (hPF) and maintained in the bioreactor until 96 hPF. In experiment 2, eggs were placed in the bioreactor immediately after they were collected and maintained in the bioreactor until 24, 36, 48, 60, 66, 72, or 96 hPF. Beginning at 96 hPF, all larvae had their vestibulo-ocular reflexes (VOR) evaluated once each day for 5 days. Only larvae that hatched from eggs that were placed in the bioreactor before 30 hPF in experiment 1 or removed from the bioreactor later than 66 hPF in experiment 2 had VOR deficits that persisted for at least 5 days. These data suggest a critical period for vestibular development in the zebrafish that begins before 30 hPF and ends after 66 hPF. To confirm this, zebrafish eggs were placed in the bioreactor at 24 hPF and removed at 72 hPF. VORs were evaluated in these larvae once each day for 5 days beginning at 96 hPF. These larvae had VOR deficits that persisted for at least 5 days. In addition, larvae that had been maintained in the bioreactor from 24 to 66 hPF or from 30 to 72 hPF, had only temporary VOR deficits. In a final experiment, zebrafish eggs were placed in the bioreactor at 3 hPF and removed at 96 hPF but the bioreactor was turned off from 24 hPF to 72 hPF. These larvae had normal VORs when they were removed from the bioreactor at 96 hPF. Taken as a whole, these data support the idea that there is a critical period for functional maturation of the zebrafish vestibular system. The developmental period identified includes the timeframe during which the vestibular primary afferent neurons are born, innervate their central and peripheral targets, and remodel their central projections.

Elevated mucosal IL-12/23p40 and IFN-gamma accompany early inflammation in IL-10-deficient (IL-10(-/-)) mice and then later decline while inflammation persists. This report addresses whether this cytokine profile reflects disease progression or inherent, age-related changes in mucosal immunity. IL-10(-/-) and wild-type (WT) mice were maintained in an ultrabarrier facility or transferred to conventional housing at 3, 12, or 30 weeks of age. Weight, stool changes, and histologic features were followed. Lamina propria mononuclear cells were cultured for cytokine analysis by ELISA. Ultrabarrier-housed IL-10(-/-) mice are statistically indistinguishable from WT mice by weight, disease activity index, and histologic inflammation. IL-10(-/-) mice but not WT, transferred at 3 weeks, develop colitis gradually, reaching a significant, sustained maximum by 15 weeks of age. Transfer at 12 weeks induces rapid disease onset in both strains, maximal at 15 weeks of age. Inflammation persists in IL-10(-/-), and WT recover. IL-10(-/-) and WT mice transferred at 30 weeks demonstrate transient diarrhea and weight loss but no chronic inflammation. Probiotics delay symptom onset only in the 12-week-old group. IFN-gamma production from ultrabarrier-housed IL-10(-/-) mice is elevated at 12 weeks of age, and older animals have decreased IFN-gamma and increased IL-4. IL-10 is important for suppressing inflammation after transfer at 3 weeks of age and limiting inflammation after transfer at 12 weeks but has little influence at 30 weeks of age. Colitis onset, progression, and response to probiotic therapy vary with immune system age, suggesting that a distinct, Th1-driven, age-dependent cytokine profile may contribute to increased colitis susceptibility in otherwise healthy mice.